A-beta immunogenic peptide carrier conjugates and methods of producing same

ABSTRACT

The present invention is directed to methods of producing conjugates of Aβ peptide immunogens with protein/polypeptide carrier molecules, which are useful as immunogens, wherein peptide immunogens are conjugated to protein carriers via activated functional groups on amino acid residues of the carrier or of the optionally attached linker molecule, and wherein any unconjugated reactive functional groups on amino acid residues are inactivated via capping, thus retaining the immunological functionality of the carrier molecule, but reducing the propensity for undesirable reactions that could render the conjugate less safe or effective. Furthermore, the invention also relates to such immunogenic products and immunogenic compositions containing such immunogenic products made by such methods.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 60/530,481, filed Dec. 17, 2003,which is incorporated herein by reference in its entirety for allpurposes

BACKGROUND OF THE INVENTION

The essence of adaptive immunity is the ability of an organism to reactto the presence of foreign substances and produce components (antibodiesand cells) capable of specifically interacting with and protecting thehost from their invasion. An “antigen” or “immunogen” is a substancethat is able to elicit this type of immune response and also is capableof interacting with the sensitized cells and antibodies that aremanufactured against it.

Antigens or immunogens are usually macromolecules that contain distinctantigenic sites or “epitopes” that are recognized and interact with thevarious components of the immune system. They can exist as individualmolecules composed of synthetic organic chemicals, proteins,lipoproteins, glycoproteins, RNA, DNA, or polysaccharides, or they maybe parts of cellular structures (bacteria or fungi) or viruses (Harlowand Lane 1988a, b, c; Male et al., 1987).

Small molecules like short peptides, although normally able to interactwith the products of an immune response, often cannot cause a responseon their own. These peptide immunogens or “haptens” as they are alsocalled, are actually incomplete antigens, and, although not able bythemselves to cause immunogenicity or to elicit antibody production, canbe made immunogenic by coupling them to a suitable carrier. Carrierstypically are protein antigens of higher molecular weight that are ableto cause an immunological response when administered in vivo.

In an immune response, antibodies are produced and secreted by theB-lymphocytes in conjunction with the T-helper (T_(H)) cells. In themajority of hapten-carrier systems, the B cells produce antibodies thatare specific for both the hapten and the carrier. In these cases, the Tlymphocytes will have specific binding domains on the carrier, but willnot recognize the hapten alone. In a kind of synergism, the B and Tcells cooperate to induce a hapten-specific antibody response. Aftersuch an immune response has taken place, if the host is subsequentlychallenged with only the hapten, usually it will respond by producinghapten-specific antibodies from memory cells formed after the initialimmunization.

Synthetic haptens mimicking some critical epitopic structures on largermacromolecules are often conjugated to carriers to create an immuneresponse to the larger “parent” molecule. For instance, short peptidesegments can be synthesized from the known sequence of a protein andcoupled to a carrier to induce immunogenicity toward the native protein.This type of synthetic approach to the immunogen production has becomethe basis of much of the current research into the creation of vaccines.However, in many instances, merely creating a B-cell response by usingsynthetic peptide-carrier conjugates, however well designed, will notalways guarantee complete protective immunity toward an intact antigen.The immune response generated by a short peptide epitope from a largerviral particle or bacterial cell may only be sufficient to generatememory at the B cell level. In these cases it is generally now acceptedthat a cytotoxic T-cell response is a more important indicator ofprotective immunity. Designing peptide immunogens with the properepitopic binding sites for both B-cell and T-cell recognition is one ofthe most challenging research areas in immunology today.

The approach to increasing immunogenicity of small or poorly immunogenicmolecules by conjugating these molecules to large “carrier” moleculeshas been utilized successfully for decades (see, e.g., Goebel et al.(1939) J. Exp. Med. 69: 53). For example, many immunogenic compositionshave been described in which purified capsular polymers have beenconjugated to carrier proteins to create more effective immunogeniccompositions by exploiting this “carrier effect.” Schneerson et al.(1984) Infect. Immun. 45: 582-591). Conjugation has also been shown tobypass the poor antibody response usually observed in infants whenimmunized with a free polysaccharide (Anderson et al. (1985) J. Pediatr.107: 346; Insel et al. (1986) J. Exp. Med. 158: 294).

Hapten-carrier conjugates have been successfully generated using variouscross-linking/coupling reagents such as homobifunctional,heterobifunctional, or zero-length cross linkers. Many such methods arecurrently available for coupling of saccharides, proteins, and peptidesto peptide carriers. Most methods create amine, amide, urethane,isothiourea, or disulfide bonds, or in some cases thioethers. Adisadvantage to the use of coupling reagents, which introduce reactivesites in to the side chains of reactive amino acid molecules on carrierand/or hapten molecules, is that the reactive sites if not neutralizedare free to react with any unwanted molecule either in vitro (thusadversely affecting the functionality or stability of the conjugate(s))or in vivo (thus posing a potential risk of adverse events in persons oranimals immunized with the preparations). Such excess reactive sites canbe reacted or “capped”, so as to inactivate these sites, utilizingvarious known chemical reactions, but these reactions may be otherwisedisruptive to the functionality of the conjugates. This may beparticularly problematic when attempting to create a conjugate byintroducing the reactive sites into the carrier molecule, as its largersize and more complex structure (relative to the hapten) may render itmore vulnerable to the disruptive effects of chemical treatment. Infact, no examples are known of methods whereby a conjugate is made byfirst activating the carrier, then reacting with the hapten in aconjugation reaction, and finally “capping” the remaining reactivesites, while preserving the ability of the resulting conjugate tofunction as an immunogenic composition having the desired properties ofthe “carrier effect”.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods of producing an immunogenicconjugate of a peptide immunogen comprising Aβ peptide or fragments ofAβ or analogs thereof with a protein/polypeptide carrier, wherein the Aβpeptide or fragments of Aβ or analogs thereof is conjugated to thecarrier via derivatized functional groups of amino acid residues of thecarrier such as lysine residues, and wherein any unconjugated,derivatized functional groups of the amino acid residues are inactivatedvia capping to block them from reacting with other molecules, includingproteins/polypeptides thereby preserving the functionality of thecarrier, such that it retains its ability to elicit the desired immuneresponses against the peptide immunogen that would otherwise not occurwithout a carrier. Furthermore, the invention also relates to conjugatesproduced by the above methods, and to immunogenic compositionscontaining such conjugates.

In one embodiment, the invention is directed to a first method forconjugating a peptide immunogen comprising Aβ peptide or fragments of Aβor analogs thereof via a reactive group of an amino acid residue of thepeptide immunogen to a protein/polypeptide carrier having one or morefunctional groups, the method comprising the steps of: (a) derivatizingone or more of the functional groups of the protein/polypeptide carrierto generate a derivatized molecule with reactive sites; (b) reacting thederivatized protein/polypeptide carrier of step (a) with a reactivegroup of an amino acid residue of the peptide immunogen under reactionconditions such that the peptide immunogen is conjugated to thederivatized protein/polypeptide carrier via the functional groups; and(c) further reacting the conjugate with a capping reagent to inactivatefree, reactive functional groups on the activated protein/polypeptidecarrier, thereby preserving the functionality of the carrier such thatit retains its ability to elicit the desired immune responses againstthe peptide immunogen that would otherwise not occur without a carrier.

In one embodiment, the protein/polypeptide carrier is selected from thegroup consisting of human serum albumin, keyhole limpet hemocyanin,immunoglobulin molecules, thyroglobulin, ovalbumin, influenzahemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malariacircumsporozite (CS) protein, hepatitis B surface antigen(HB_(s)Ag₁₉₋₂₈, Heat Shock Protein (HSP) 65, Bacillus Calmette-Guerin(BCG), cholera toxin, cholera toxin mutants with reduced toxicity,diphtheria toxin, CRM₁₉₇ protein that is cross-reactive with diphtheriatoxin, recombinant Streptococcal C5a peptidase, Streptococcus pyogenesORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF2452, Streptococcus pneumoniae pneumolysin, pneumolysin mutants withreduced toxicity, Chlamydia pneumoniae ORF T367, Chlamydia pneumoniaeORF T858, Tetanus toxoid, HIV gp120 T1, microbial surface componentsrecognizing adhesive matrix molecules (MSCRAMMS), growth factor/hormone,cytokines and chemokines.

In another embodiment, the protein/polypeptide carrier contains a T-cellepitope.

In yet another embodiment, the protein/polypeptide carrier is abacterial toxoid such as a tetanus toxoid, cholera toxin or choleratoxin mutant as described above. In a preferred embodiment, theprotein/polypeptide carrier is CRM₁₉₇.

In still yet another embodiment, the protein/polypeptide carrier may bean influenza hemagglutinin, a PADRE polypeptide, a malaria CS protein, aHepatitis B surface antigen (HSBAG₁₉₋₂₈), a heat shock protein 65 (HSP65), or a polypeptide from Mycobacterium tuberculosis (BCG).

In a preferred embodiment, the protein/polypeptide carrier is selectedfrom Streptococcal rC5a peptidase, Streptococcus pyogenes ORF1224,Streptococcus pyogenes ORF1664 or Streptococcus pyogenes ORF2452,Streptococcus pneumoniae pneumolysin, pneumolysin mutants with reducedtoxicity, Chlamydia pneumoniae ORF T367, and Chlamydia pneumoniae ORFT858.

In one embodiment, protein/polypeptide carrier is a growth factor orhormone, which stimulates or enhances immune response and is selectedfrom the group consisting of IL-1, IL-2, γ-interferon, IL-10, GM-CSF,MIP-1α, MIP-1β, and RANTES.

In one aspect, the invention provides a peptide immunogen comprising Aβpeptide or fragments of Aβ or analogs thereof eliciting an immunogenicresponse against certain epitopes within Aβ. Immunogenic peptides of theinvention include immunogenic heterologous peptides. In some immunogenicpeptides, an Aβ fragment is linked to a carrier to form an immunogenicheterologous peptide, and then this heterologous peptide is linked to acarrier using a method of the present invention to form a conjugate.

In another aspect of the invention, the peptide immunogen is apolypeptide comprising an N-terminal segment of at least residues 1-5 ofAβ, the first residue of Aβ being the N-terminal residue of thepolypeptide, wherein the polypeptide is free of a C-terminal segment ofAβ. In yet another aspect of the invention, the peptide immunogen is apolypeptide comprising an N-terminal segment of Aβ, the segmentbeginning at residue 1-3 of Aβ and ending at residues 7-11 of Aβ. Insome aspects of the invention, the peptide immunogen is an agent thatinduces an immunogenic response against an N-terminal segment of Aβ, thesegment beginning at residue 1-3 of Aβ and ending at residues 7-11 of Aβwithout inducing an immunogenic response against an epitope withinresidues 12-43 of Aβ43. In another aspect of the invention, the peptideimmunogen is a heterologous polypeptide comprising a segment of Aβlinked to a heterologous amino acid sequence that induces a helperT-cell response against the heterologous amino acid sequence and therebya B-cell response against the N-terminal segment.

In some peptide immunogens, the N-terminal segment of Aβ is linked atits C-terminus to a heterologous polypeptide. In some peptideimmunogens, the N-terminal segment of Aβ is linked at its N-terminus toa heterologous polypeptide. In some peptide immunogens, the N-terminalsegment of Aβ is linked at its N and C termini to first and secondheterologous polypeptides. In some peptide immunogens, the N-terminalsegment of Aβ is linked at its N terminus to a heterologous polypeptide,and at its C-terminus to at least one additional copy of the N-terminalsegment. In some peptide immunogens, the polypeptide comprises fromN-terminus to C-terminus, the N-terminal segment of Aβ, a plurality ofadditional copies of the N-terminal segment, and the heterologous aminoacid segment.

In some of the above peptide immunogens, the polypeptide furthercomprises at least one additional copy of the N-terminal segment. Insome of the above peptide immunogens, the fragment is free of at leastthe 5 C-terminal amino acids in Aβ43.

In some aspects of the above peptide immunogens, the fragment comprisesup to 10 contiguous amino acids from Aβ.

In another aspect, the invention provides a peptide immunogen comprisingAβ peptide or fragments of Aβ or analogs thereof eliciting animmunogenic response against certain epitopes within Aβ may be in aconfiguration referred to as a multiple antigenic peptide (MAP)configuration.

In some of the above aspects of the invention, the peptide immunogenfrom the N-terminal half of Aβ. In some aspects of the invention, thepeptide immunogen is an Aβ fragment selected from the group consistingof Aβ1-3, 1-4, 1-5, 1-6, 1-7, 1-10, 1-11, 1-12, 1-16, 3-6, and 3-7. Insome of the above aspects of the invention, the peptide immunogen isfrom the internal region of Aβ. In some aspects of the invention, thepeptide immunogen is an Aβ fragment selected from the group consistingof Aβ13-28, 15-24, 17-28, and 25-35. In some of the above aspects of theinvention, the peptide immunogen from the C-terminal end of Aβ. In someaspects of the invention, the peptide immunogen is an Aβ fragmentselected from the group consisting of Aβ33-42, 35-40, and 35-42. In someaspects of the invention, the peptide immunogen is an Aβ fragmentselected from the group consisting of Aβ1-3, 1-4, 1-5, 1-6, 1-7, 1-10,1-11, 1-12, 1-16, 1-28, 3-6, 3-7, 13-28, 15-24, 17-28, 25-35, 33-42,35-40, and 35-42. In some aspects of the invention, the peptideimmunogen is an Aβ fragment selected from the group consisting of Aβ1-5,Aβ1-7, Aβ1-9, and Aβ1-12. In some aspects of the invention, the peptideimmunogen is an Aβ fragment selected from the group consisting ofAβ1-5-L, Aβ1-7-L, Aβ1-9-L, and Aβ1-12-L, where L is a linker. In someaspects of the invention, the peptide immunogen is an Aβ fragmentselected from the group consisting of Aβ1-5-L-C, Aβ1-7-L-C, Aβ1-9-L-C,and Aβ1-12-L-C, where C is a cysteine amino acid residue.

In some aspects of the invention, the peptide immunogen is an Aβfragment selected from the group consisting of Aβ16-22, Aβ16-23,Aβ17-23, Aβ17-24, Aβ18-24, and Aβ18-25. In some aspects of theinvention, the peptide immunogen is an A: fragment selected from thegroup consisting of Aβ16-22-C, Aβ16-23-C, Aβ17-23-C, Aβ17-24-C,Aβ18-24-C, and Aβ18-25-C, where C is a cysteine amino acid residue. Inother aspects of the invention, the peptide immunogen is an Aβ fragmentselected from the group consisting of C-Aβ16-22, C-Aβ16-23, C-Aβ17-23,C-Aβ17-24, C-Aβ18-24, and C-Aβ18-25, where C is a cysteine amino acidresidue.

In some of the above peptide immunogens, the heterologous polypeptide isselected from the group consisting of peptides having a T-cell epitope,a B-cell epitope and combinations thereof.

In one embodiment, the functional group of one or more amino acidmolecules of the protein/polypeptide carrier or of the optionallyattached polypeptide linker is derivatized using a cross-linkingreagent. In another embodiment, the derivatizing reagent is azero-length cross-linking reagent. In another embodiment, thederivatizing reagent is a homobifunctional cross-linking reagent. In yetanother embodiment, the derivatizing reagent is a heterobifunctionalcross-linking reagent.

In a preferred embodiment, the heterobifunctional reagent is a reagentthat reacts with a primary or a ε-amine functional group of one or moreamino acid molecules of the protein/polypeptide carrier and a pendantthiol group of one or more amino acid molecules of the peptideimmunogen. In one embodiment, the heterobifunctional reagent isN-succinimidyl bromoacetate.

In another embodiment, the primary or ε-amine functional group islysine. In yet another embodiment, the derivatization of the primary orε-amine functional group of the lysine of the protein/polypeptidecarrier with N-succinimidyl bromoacetate results in the bromoacetylationof the primary or ε-amine residues on lysine molecules on theprotein/polypeptide carrier. In a more preferred embodiment, the pendantthiol group is a cysteine residue of the peptide immunogen, which may belocalized at the amino-terminus of the peptide immunogen, at thecarboxy-terminus of the peptide immunogen or internally in the peptideimmunogen.

In another embodiment, the pendant thiol group is generated by athiolating reagent such as N-acetyl homocysteinethio lactone, Traut'sreagent (2-iminothilane) SATA (N-Succinimidyl S-acetylthioacetate), SMPT(4-Succinimidyloxycarbonyl-methyl2-pyridyldithio toluene), Sulfo LC SPDP(Sulfo Succinimidyl pyridyl dithio propionamido hexanoate), SPDP(Succinimidyl pyridyl dithio propionate). In a preferred embodiment, thecapping reagent that is used to inactivate free reactive, functionalgroups on the activated protein/polypeptide carrier is selected from thereagent group consisting of cysteamine, N-acetylcysteamine, andethanolamine.

In a particularly preferred embodiment, the capping reagent that is usedto inactivate free reactive functional groups on the activatedprotein/polypeptide carrier is selected from the reagent groupconsisting of sodium hydroxide, sodium carbonate, ammonium bicarbonateand ammonia.

In one embodiment, the reactive group of the amino acid residue of thepeptide immunogen is a free sulfhydryl group.

In another embodiment, one or more of the functional groups are on alinker, which is optionally attached to the protein/polypeptide carrier.In a preferred embodiment, the linker is a peptide linker. In a morepreferred embodiment, the peptide linker is polylysine.

In another embodiment, the invention is directed to a second method forconjugating a peptide immunogen comprising Aβ peptide or fragments of Aβor analogs thereof Aβ or analogs thereof with a protein/polypeptidecarrier having the structure:

wherein,

C is a protein/polypeptide carrier and X is a derivatizable functionalgroup of an amino acid residue on the protein/polypeptide carrier oroptionally of an amino acid residue of a peptide linker covalentlyattached to the protein/polypeptide carrier, and wherein m is an integergreater than 0, but less than or equal to 85, the method comprising thesteps of: (a) derivatizing one or more of the functional groups of theprotein/polypeptide carrier or of the optionally attached linkermolecule to generate a derivatized molecule with reactive sites; (b)reacting the derivatized protein/polypeptide carrier of step (a) with areactive group of an amino acid residue of the peptide immunogen to forma covalently coupled peptide immunogen-protein/polypeptide carrierconjugate; and (c) further reacting the said conjugate with a cappingreagent to inactive the free reactive functional groups on the activatedprotein/polypeptide carrier, such that the capped groups are not free toreact with other molecules, including proteins/polypeptides therebypreserving the functionality of the carrier, such that it retains itsability to elicit the desired immune responses against the peptideimmunogen that would otherwise not occur without a carrier so as togenerate a capped peptide immunogen-protein/polypeptide carrierconjugate having the formula:

wherein,

C is the protein/polypeptide carrier and X^(d) is a derivatizedfunctional group of an amino acid residue of the protein/polypeptidecarrier or optionally of an amino acid residue of a peptide linkercovalently attached to the protein/polypeptide carrier, and, wherein,

P is the peptide immunogen molecule covalently attached to thederivatized functional group on the amino acid residue on the proteincarrier or optionally on an amino acid residue on a peptide linkercovalently attached to a protein/polypeptide carrier, R is a cappingmolecule covalently attached to the derivatized functional group on anamino acid residue on the protein/polypeptide carrier or optionally onan amino acid residue on a peptide linker covalently attached to aprotein/polypeptide carrier, n is an integer greater than 0, but lessthan or equal to 85, and p is an integer greater than 0, but less than85.

The detailed embodiments for the first method described above are alsoapplicable to the conjugates just described prepared by the secondmethod.

In one embodiment, the invention is directed to peptideimmunogen-comprising Aβ peptide or fragments for Aβ or analogsthereof/polypeptide carrier conjugates wherein the protein/polypeptidecarrier has the formula:

wherein,

C is a protein/polypeptide carrier and X is a derivatizable functionalgroup of an amino acid residue on the protein/polypeptide carrier oroptionally of an amino acid residue of a peptide linker covalentlyattached to the protein/polypeptide carrier, and, wherein, m is aninteger greater than 0, but less than or equal to 85, and wherein thecapped peptide immunogen-protein/polypeptide carrier conjugate has theformula:

wherein,

C is the protein/polypeptide carrier and X^(d) is a derivatizedfunctional group of an amino acid residue of the protein/polypeptidecarrier or optionally of an amino acid residue of a peptide linkercovalently attached to the protein/polypeptide carrier, and, wherein, Pis the peptide immunogen molecule covalently attached to the derivatizedfunctional group of the amino acid residue of the protein carrier oroptionally of an amino acid residue of a peptide linker covalentlyattached to a protein/polypeptide carrier, R is a capping moleculecovalently attached to the derivatized functional group of an amino acidresidue of the protein/polypeptide carrier or optionally of an aminoacid residue of a peptide linker covalently attached to aprotein/polypeptide carrier, thereby preserving the functionality of thecarrier, such that it retains its ability to elicit the desired immuneresponses against the peptide immunogen that would otherwise not occurwithout a carrier, n is an integer greater than 0, but less than orequal to 85, and p is an integer greater than 0, but less than 85.

The detailed embodiments for the first and second methods describedabove are also applicable to the conjugates just described.

In another embodiment, the invention is directed to peptideimmunogen-comprising Aβ peptide or fragments of Aβ or analogs thereofpolypeptide carrier conjugates generated according to the second methodof the invention and having the formula:

wherein,

C is the protein/polypeptide carrier and X^(d) is a derivatizedfunctional group of an amino acid residue of the protein/polypeptidecarrier or optionally of an amino acid residue of a peptide linkercovalently attached to the protein/polypeptide carrier, and, wherein, Pis the peptide immunogen molecule covalently attached to the derivatizedfunctional group of the amino acid residue of the protein carrier oroptionally of an amino acid residue of a peptide linker covalentlyattached to a protein/polypeptide carrier, R is a capping moleculecovalently attached to the derivatized functional group of an amino acidresidue of the protein/polypeptide carrier or optionally of an aminoacid residue of a peptide linker covalently attached to aprotein/polypeptide carrier thereby preserving the functionality of thecarrier, such that it retains its ability to elicit the desired immuneresponses against the peptide immunogen that would otherwise not occurwithout a carrier, n is an integer greater than 0, but less than orequal to 85, and p is an integer greater than 0, but less than 85.

The detailed embodiments for the second method described above are alsoapplicable to the conjugates generated by the second method, as justdescribed.

In another embodiment, the invention is directed to immunogeniccompositions comprising a conjugate of a peptide immunogen with aprotein/polypeptide carrier generated by the second method of theinvention, together with one or more pharmaceutically acceptableexcipients, diluents, and adjuvants.

The detailed embodiments for the second method and the conjugatesgenerated thereby described above are also applicable to immunogeniccompositions containing those conjugates as just described.

In another embodiment, the invention is directed to a method forinducing an immune response in a mammalian subject, which comprisesadministering an effective amount of an immunogenic composition of thepresent invention to the subject.

The detailed embodiments applicable to the immunogenic compositioncontaining the conjugates of the present invention are also applicableto the embodiment of the invention directed to the method of use ofthese immunogenic compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Flow chart depicting the process chemistry used for conjugationof Aβ peptide fragments to protein/polypeptide carrier CRM₁₉₇ to formthe Aβ/CRM₁₉₇ conjugate.

FIG. 2: Flow chart depicting acid hydrolysis chemistry used forquantitative determination of S-carboxymethylcysteine andS-carboxymethylcysteamine as evaluation of the degree of conjugation ofpeptide immunogen-protein/polypeptide conjugates such as the Aβ/CRM₁₉₇conjugate.

FIG. 3: This figure depicts the pH dependence of the Aβ peptide/CRMconjugation reaction.

FIG. 4: This figure depicts the dependence of Aβ-peptide/CRM conjugationon peptide:CRM ratio.

FIG. 5: Verification of capping process for Aβ1-7/CRM conjugation. ThepH of the reaction was 9.15. Reaction time with peptide was 16 hrs,capping with N-acetylcysteamine was 8 hrs.

FIG. 6: Conjugation and capping with various peptide: CRM ratios withpeptide. The pH of the reaction was 9.0. Reaction time with peptide was16 hrs, capping with N-acetylcysteamine was 8 hrs.

FIG. 7: Day 36 titers of primate sera following immunization of primateswith Aβ peptide conjugates with various adjuvants.

FIG. 8: Day 64 titers of primate sera following immunization of primateswith Aβ-peptide conjugates with various adjuvants.

FIG. 9: Primate titers by day and treatment group. Primates wereimmunized with Aβ1-7 or Aβ1-5 CRM₁₉₇ conjugates with alum or RC529 asadjuvants and titers of anti-AA antibodies were measured at day 29, 36,57 and 54.

FIG. 10: Peptide-protein conjugates were characterized using SDS-PAGEWestern blot analysis with a tris-tricine precast gel. The lanes are:marker (lane 1); L-28375 24/01 (lane 2); L-28375 24/02 (lane 3); L-2837524/03 (lane 4); L-28375 24/04 (lane 5); L-28375 24/05 (lane 6); L-2837524/06 (lane 7) L-28375 24/07 (lane 8); L-28375 24/08 (lane 9); L-2837524/09 (Mock) (lane 10); and, BrAcCRM₁₉₇ (lane 11).

BRIEF DESCRIPTION OF SEQUENCES SEQ ID NO: Sequence Description  1DAEFR-C Aβ1-5-C  2 DAEFRHD-C Aβ1-7-C  3 DAEFRHDSG-C Aβ1-9-C  4DAEFRHDSGYEV-C Aβ1-12-C  5 DAEFR-GAGA-C Aβ1-5-L-C  6 DAEFRHD-GAGA-CAβ1-7-L-C  7 DAEFRHDSG-GAGA-C Aβ1-9-L-C  8 DAEFRHDSGYEV-GAGA-CAβ1-12-L-C  9 VEYGSDHRFEAD-C Aβ12-1-C 10 GAGA Linker peptide 11PKYVKQNTLKLAT Influenza Hemagglutinin: HA₃₀₇₋₃₁₉ 12 AKXVAAWTLKAAA PAN-DRPeptide (PADRE peptide) 13 EKKIAKMEKASSVFNV Malaria CS: T3 epitope 14FELLTRILTI Hepatitis B surface antigen: HB_(s)Ag₁₉₋₂₈ 15DQSIGDLIAEAMDKVGNEG Heat Shock Protein 65: hsp65₁₅₃₋₁₇₁ 16QVHFQPLPPAVVKL Bacillus Calmette-Guerin (BCG) 17 QYIKANSKFIGITEL Tetanustoxoid: TT₈₃₀₋₈₄₄ 18 FNNFTVSFWLRVPKVSASHLE Tetanus toxoid: TT₉₄₇₋₉₆₇ 19KQIINMWQEVGKAMY HIV gp120 T1 20 DAEFRHD-QYIKANSKFIGITEL-C-Aβ₁₋₇/TT₈₃₀₋₈₄₄/C/TT₉₄₇₋₉₆₇/Aβ₁₋₇ FNKFTVSFWLRVPKVSASHLE- DAEFRHD 21DAEFRHDSGYEVHHQKLVFFAEDVGSN Aβ₁₋₄₂ KGAIIGLMVGGVVIA 22DAEFRHDQYIKANSKFIGITEL AN90549: Aβ₁₋₇/TT₈₃₀₋₈₄₄ (used in a MAP4configuration) 23 DAEFRHDFNNFTVSFWLRVPKVSASHLE AN90550: Aβ₁₋₇/TT₉₄₇₋₉₆₇(used in a MAP4 configuration) 24 DAEFRHD- AN90542: Aβ₁₋₇/TT₈₃₀₋₈₄₄+ TT₉₄₇₋₉₆₇ QYIIKANSKFIGITELFNNFTVSFWLRVPK (used in a linearconfiguration) VSASHLE 25 EFRHDSG-QYIKANSKFIGITEL AN90576:Aβ₃₋₉/TT₂₃₀₋₈₄₄ (used in a MAP4 configuration) 26 AKXVAAWTLKAAA-DAEFRHDAN90562: Aβ₁₋₇/PADRE 27 DAEFRHD-DAEFRHDD- AN90543: Aβ₁₋₇ × 3/PADREAEFRHDAKXVAAWTLKAAA 28 AKXVAAWTLKAAA-DAEFRHD- PADRE/Aβ₁₋₇ × 3DAEFRHD-DAEFRHD 29 DAEFRHD-AKXVAAWTLKAAA Aβ₁₋₇ × 3/PADRE 30DAEFRHD-ISQAVHAAHAEINEAGR Aβ₁₋₇/albumin fragment 31FRHDSGY-ISQAVHAAHAEINEAGR Aβ₄₋₁₀/albumin fragment 32EFRHDSG-ISQAVHAAHAEINEAGR Aβ₃₋₉/albumin fragment 33PKYVKQNTLKLAT-DAEFRHD- HA₃₀₇₋₃₁₉/Aβ₁₋₇ × 3 DAEFRHD-DAEFRHD 34DAEFRHD-PKYVKQNTLKLAT- Aβ₁₋₇/HA₃₀₇₋₃₁₉/Aβ₁₋₇ DAEFRHD 35DAEFRHD-DAEFRHD-DAEFRHD- Aβ₁₋₇ × 3/HA₃₀₇₋₃₁₉ PKYVKQNTLKLAT 36DAEFRHD-DAEFRHD- Aβ₁₋₇ × 2/HA₃₀₇₋₃₁₉ PKYVKQNTLKLAT 37DAEFRHD-PKYVKQNTLKLAT- Aβ₁₋₇/HA₃₀₇₋₃₁₉/Malaria CS/ EKKIAKMEKASSVFNV-TT₈₃₀₋₈₄₄/TT₉₄₇₋₉₆₇/Aβ₁₋₇ QYIKANSKFIGITEL- FNNFTVSFWLRVPKVSASHLE-DAEFRHD 38 DAEFRHD-DAEFRHD-DAEFRHD- Aβ₁₋₇ × 3/TT₈₃₀₋₈₄₄/C/TT₉₄₇₋₉₆₇QYIKANSKFIGITEL-C- FNNFTVSFWLRVPKVSASHLE 39 DAEFRHD-QYIKANSKFIGITEL-C-Aβ₁₋₇/TT₈₃₀₋₈₄₄/C/TT₉₄₇₋₉₆₇ FNXFTVSFWLRVPKVSASHLE 40GADDVVDSSKSFVMENFSSYHGTKPGY CRMβ₁₉₇ VDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVV KVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLS LPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVR RSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEE FHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAAL SILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFV ESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDI KITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSP VYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEI KS 41 ISQAVHAAHAEINEAGR Albumin fragment42 DAEFGHDSGFEVRHQKLVFFAEDVGSNKG Murine A(1-42 AIIGLMVGGVVIA 43VFFAEDVG-C A(18-25 -C 44 LVFFAEDV-C A(17-24 -C 45 KLVFFAED-C A(16-23 -C46 C-VFFAEDVG C-A(18-25 47 C-LVFFAEDV C-A(17-24 48 C-KLVFFAED C-A(16-2349 VFFAEDV-C A(18-24 -C 50 LVFFAED-C A(17-23 -C 51 KLVFFAE-C A(16-22 -C52 C-VFFAEDV C-Aβ₁₈₋₂₄ 53 C-LVFFAED C-Aβ₁₇₋₂₃ 54 C-KLVFFAE C-Aβ₁₆₋₂₂

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods of generating peptideimmunogen-carrier conjugates wherein the unreacted active functionalgroups on the carrier which are generated during activation areinactivated by using capping reagents such as N-Acetylcysteamine inorder to prevent them from reacting further. The present invention isalso directed to capped carrier-peptide immunogen conjugates generatedby those methods and to immunogenic compositions comprising saidconjugates.

The approach of increasing immunogenicity of small or poorly immunogenicmolecules, such as saccharides, through conjugation has been utilizedsuccessfully for decades (see, e.g., Goebel et al. (1939) J. Exp. Med.69: 53), and many immunogenic compositions have been described in whichpurified capsular polymers have been conjugated to carrier proteins tocreate more effective immunogenic compositions by exploiting this“carrier effect”. For example, Schneerson et al. (J. Exp. Med. 152:361-376, 1980), describe Haemophilus influenzae b polysaccharide proteinconjugates that confer immunity to invasive diseases caused by thatmicroorganism. Conjugates of PRP (polyribosylribitol phosphate, acapsular polymer of H. influenzae b) have been shown to be moreeffective than immunogenic compositions based on the polysaccharidealone (Chu et al., (1983) Infect. Immun. 40: 245; Schneerson et al.(1984), Infect. Immun. 45: 582-591). Conjugation has also been shown tobypass the poor antibody response usually observed in infants whenimmunized with a free polysaccharide (Anderson et al. (1985) J. Pediatr.107: 346; Insel et al. (1986) J. Exp. Med. 158: 294).

A further advantage of using as the protein carrier a bacterial toxin ortoxoid against which routine immunization of humans (e.g., tetanus ordiphtheria) is a standard practice is that a desired immunity to thetoxin or toxoid is induced along with immunity against the pathogensassociated with the capsular polymer.

Antigenic determinant/hapten-carrier conjugates also are being used toproduce highly specific monoclonal antibodies that can recognizediscrete chemical epitopes on the coupled hapten. The resultingmonoclonals often are used to investigate the epitopic structure andinteractions between native proteins. In many cases, the antigenicdeterminants/haptens used to generate these monoclonals are smallpeptide segments representing crucial antigenic sites on the surface oflarger proteins. The criteria for a successful carrier to be used ingenerating an antigenic determinant/hapten-carrier conjugate are thepotential for immunogenicity, the presence of suitable functional groupsfor conjugation with an antigenic determinant/hapten, reasonablesolubility properties even after derivatization and lack of toxicity invivo.

These criteria are met by the conjugates generated by the methods of theinstant invention. The conjugates may be any stable peptideimmunogen-carrier conjugates generated using the conjugation processdescribed herein. The conjugates are generated using a process of theinstant invention wherein a protein/polypeptide carrier having thefollowing structure:

is covalently attached to a protein/polypeptide carrier,wherein,

C is a protein/polypeptide carrier and X is a derivatizable functionalgroup on an amino acid residue on the protein/polypeptide carrier oroptionally on an amino acid residue on a peptide linker covalentlyattached to the protein/polypeptide carrier, and wherein m is an integergreater than 0, but less than or equal to 85, is covalently attached toa peptide immunogen and wherein the peptideimmunogen-protein/polypeptide carrier conjugate has the followingformula, is represented by the following formula:

wherein,

C is the protein/polypeptide carrier and X^(d) is a derivatizedfunctional group on an amino acid residue on the protein/polypeptidecarrier or optionally on an amino acid residue on a peptide linkercovalently attached to the protein/polypeptide carrier, P is a peptideimmunogen covalently attached to the derivatized functional group on theamino acid residue on the protein/polypeptide carrier or optionally onan amino acid residue on a peptide linker covalently attached to aprotein/polypeptide carrier, R is a capping molecule covalently attachedto the derivatized functional group on an amino acid residue on theprotein/polypeptide carrier or optionally on an amino acid residue on apeptide linker covalently attached to a protein/polypeptide carrierthereby preserving the functionality of the carrier, such that itretains its ability to elicit the desired immune responses against thepeptide immunogen that would otherwise not occur without a carrier, n isan integer greater than 0, but less than or equal to 85, and p is aninteger greater than 0, but less than 85.

Selection of Carriers

Some peptide immunogens contain the appropriate epitope for inducing animmune response, but are too small to be immunogenic. In this situation,the peptide immunogens are linked to a suitable carrier to help elicitan immune response. In the above schematic representation of the peptideimmunogens-carrier conjugate generated by a process of the presentinvention, C is a protein/polypeptide carrier to which peptideimmunogens are conjugated directly via derivatized functional groups onamino acid residues on the carrier themselves or indirectly viaderivatized functional groups on peptide linkers covalently attached tothe carriers. Suitable protein/polypeptide carriers include, but are notlimited to, albumin (including humanserum albumin), keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin,MSCRAMMS, tetanus toxoid, or a toxoid from other pathogenic bacteriahaving reduced toxicity, including mutants, such as diphtheria, E. coli,cholera, or H. pylori or an attenuated toxin derivative. One suchcarrier is the CRM₁₉₇ protein (SEQ ID NO.:40) that is cross-reactivewith diphtheria toxin.

Other carriers include T-cell epitopes that bind to multiple MHCalleles, e.g., at least 75% of all human MHC alleles. Such carriers aresometimes known in the art as “universal T-cell epitopes.” Exemplarycarriers with universal T-cell epitopes include:

Influenza Hemagglutinin: PKYVKQNTLKLAT HA₃₀₇₋₃₁₉ (SEQ. ID NO.11) PAN-DRPeptide AKXVAAWTLKAAA (PADRE peptide) (SEQ. ID NO.12) Malaria CS: T3epitope EKKIAKMEKASSVFNN (SEQ. ID NO.13) Hepatitis B surface antigen:FELLTRILTI HB_(s)Ag₁₉₋₂₈ (SEQ. ID NO.14) Heat Shock Protein 65:QSIGDLIAEAMDKVGNEG hsp65₁₅₃₋₁₇₁ (SEQ. ID NO.15) Bacillus Calmette-GuerinQVHFQPLPPAVVKL (BCG) (SEQ. ID NO.16) Tetanus toxoid: TT₈₃₀₋₈₄₄QYIKANSKFIGITEL (SEQ. ID NO.17) Tetanus toxoid: TT₉₄₇₋₉₆₇NNFTVSFWLRVPKVSASHLE (SEQ. ID NO.18) HIV gp120 T1: KQIINMWQEVGKAMY (SEQ.ID NO.19) CRM₁₉₇ See the Brief Description of the Sequences (SEQ IDNO.:40) Albumin fragment ISQAVHAAHAEINEAGR (SEQ ID NO:41)

Other carriers for stimulating or enhancing an immune response and towhich a peptide immunogen or a hapten can be conjugated includecytokines such as IL-1, IL-1α and β peptides, IL-2, γINF, IL-10, GM-CSF,and chemokines, such as MIP 1α and β and RANTES. Immunogenic peptidescan also be linked to proteins/peptide carriers that enhance transportacross tissues, as described in O'Mahony, WO 97/17163 and WO 97/17614,which are hereby incorporated by reference in their entirety for allpurposes.

Still further carriers include recombinant Streptococcal C5a peptidase,Streptococcus pyogenes ORFs 1224, 1664 and 2452, Chlamydia pneumoniaeORFs T367 and T858, Streptococcus pneumonia pneumolysin, pneumolysinmutants with reduced toxicity, growth factors, and hormones.

In one preferred embodiment of the present invention, the carrierprotein is CRM₁₉₇, a non-toxic mutant of diphtheria toxin with one aminoacid change in its primary sequence. The glycine present at the aminoacid position 52 of the molecule is replaced with a glutamic acid due toa single nucleic acid codon change. Due to this change, the proteinlacks ADP-ribosyl transferase activity and becomes non-toxic. It has amolecular weight of 58,408 Da. CRM₁₉₇ is produced in large quantities byrecombinant expression in accordance with U.S. Pat. No. 5,614,382, whichis hereby incorporated by reference. Conjugations of saccharides as wellas peptides to CRM₁₉₇ are carried out by linking through the s-aminogroups of lysine residues. It has been well established through severalcommercial products that CRM₁₉₇ is an excellent and safe carrier forB-cell epitopes.

Immunogenic Peptides

As used herein, the term “peptide immunogen” or “hapten” is any proteinor subunit structure/fragment/analog derived therefrom that can elicit,facilitate, or be induced to produce an immune response onadministration to a mammal. In particular, the term is used to refer toa polypeptide antigenic determinant from any source (bacteria, virus oreukaryote), which may be coupled to a carrier using a method disclosedherein. Such polypeptide immunogen/antigenic determinants may be ofviral, bacterial or eukaryotic cell origin.

Peptide immunogens can be conjugated to a carrier for use as animmunotherapeutic in the prevention, treatment, prophylaxis oramelioration of various human diseases. Such peptide immunogens includethose derived from Aβ a peptide of 39-43 amino acids, preferably 42amino acids, which is the principal component of characteristic plaquesof Alzheimer's disease (AD) (see U.S. Pat. No. 4,666,829; Glenner & Wong(1984) Biochem. Biophys. Res. Commun. 120: 1131, Hardy (1984) TINS 20:1131; Hardy (1977) TINS 20: 154), those derived from amyloid peptides ofamylin, a polypeptide material produced by pancreatic islet cells thathas been implicated in type II diabetes, peptides derived from lowdensity lipoprotein gene products, which have been implicated inatherosclerosis and antigenic peptides derived from inflammatorycytokines and growth factors such as interleukin 6 (IL-6), tumornecrosis factor α (TNF-α) and GDF-8. Such eukaryotic peptide immunogensmay include either T-cell (CTL) or B-cell epitope, also known asβ-amyloid protein, or A4 peptide.

Aβ, also known as β-amyloid peptide, or A4 peptide (see U.S. Pat. No.4,666,829; Glenner & Wong, Biochem. Biophys. Res. Commun., 120, 1131(1984)), is a peptide of 39-43 amino acids, which is the principalcomponent of characteristic plaques of Alzheimer's disease. Aβ isgenerated by processing of a larger protein APP by two enzymes, termed βand γ secretases (see Hardy, TINS 20, 154 (1997)). Known mutations inAPP associated with Alzheimer's disease occur proximate to the site of βor γ secretase, or within Aβ. For example, position 717 is proximate tothe site of γ-secretase cleavage of APP in its processing to Aβ, andpositions 670/671 are proximate to the site of β-secretase cleavage. Itis believed that the mutations cause AD by interacting with the cleavagereactions by which Aβ is formed so as to increase the amount of the42/43 amino acid form of Aβ generated.

Aβ has the unusual property that it can fix and activate both classicaland alternate complement cascades. In particular, it binds to C1q andultimately to C3bi. This association facilitates binding to macrophagesleading to activation of B cells. In addition, C3bi breaks down furtherand then binds to CR2 on B cells in a T cell dependent manner leading toa 10,000-fold increase in activation of these cells. This mechanismcauses Aβ to generate an immune response in excess of that of otherantigens.

Aβ has several natural occurring forms. The human forms of Aβ arereferred to as Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43. The sequences of thesepeptides and their relationship to the APP precursor are illustrated byFIG. 1 of Hardy et al., TINS 20, 155-158 (1997). For example, Aβ42 hasthe sequence:

H₂N-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala-OH  (SEQID NO. 21).

Aβ41, Aβ40 and Aβ39 differ from Aβ42 by the omission of Ala, Ala-Ile,and Ala-Ile-Val respectively from the C-terminal end. Aβ43 differs fromAβ42 by the presence of a threonine residue at the C-terminus.

Peptide immunogens which are fragments of Aβ are advantageous relativeto the intact molecule for use in the present methods for severalreasons. First, because only certain epitopes within Aβ induce a usefulimmunogenic response for treatment of Alzheimer's disease, an equaldosage of mass of a fragment containing such epitopes provides a greatermolar concentration of the useful immunogenic epitopes than a dosage ofintact Aβ. Second, certain peptide immunogens of Aβ generate animmunogenic response against amyloid deposits without generating asignificant immunogenic response against APP protein from which Aβderives. Third, peptide immunogens of Aβ are simpler to manufacture thanintact Aβ due to their shorter size. Fourth, peptide immunogens of Aβ donot aggregate in the same manner as intact Aβ, simplifying preparationof conjugates with carriers.

Some peptide immunogens of Aβ have a sequence of at least 2, 3, 5, 6,10, or 20 contiguous amino acids from a natural peptide. Some peptideimmunogens have no more than 10, 9, 8, 7, 5 or 3 contiguous residuesfrom Aβ. In a preferred embodiment, peptide immunogens from theN-terminal half of Aβ are used for preparing conjugates. Preferredpeptide immunogens include Aβ1-5, 1-6, 1-7, 1-10, 1-11, 3-7, 1-3, and1-4. The designation Aβ1-5 for example, indicates an N-terminal fragmentincluding residues 1-5 of Aβ. Aβ fragments beginning at the N-terminusand ending at a residue within residues 7-11 of Aβ are particularlypreferred. The fragment Aβ1-12 can also be used but is less preferred.In some methods, the fragment is an N-terminal fragment other thanAβ1-10. Other preferred fragments include Aβ13-28, 15-24, 1-28, 25-35,35-40, 35-42 and other internal fragments and C-terminus fragments.

Some Aβ peptides of the invention are immunogenic peptides that onadministration to a human patient or animal generate antibodies thatspecifically bind to one or more epitopes between residues 16 and 25 ofAβ. Preferred fragments include Aβ16-22, 16-23, 17-23, 17-24, 18-24, and18-25. Antibodies specifically binding to epitopes between residues 16and 25 specifically bind to soluble Aβ without binding to plaques of Aβ.These types of antibody can specifically bind to soluble Aβ in thecirculation of a patient or animal model without specifically binding toplaques of Aβ deposits in the brain of the patient or model. Thespecific binding of antibodies to soluble Aβ inhibits the Aβ from beingincorporated into plaques thus either inhibiting development of theplaques in a patient or inhibiting a further increase in the size orfrequency of plaques if such plaques have already developed beforetreatment is administered.

Preferably, the fragment of Aβ administered lacks an epitope that wouldgenerate a T-cell response to the fragment. Generally, T-cell epitopesare greater than 10 contiguous amino acids. Therefore, preferredfragments of Aβ are of size 5-10 or preferably 7-10 contiguous aminoacids or most preferably 7 contiguous amino acids; i.e., sufficientlength to generate an antibody response without generating a T-cellresponse. Absence of T-cell epitopes is preferred because these epitopesare not needed for immunogenic activity of fragments, and may cause anundesired inflammatory response in a subset of patients (Anderson etal., (2002) J. Immunol. 168, 3697-3701; Senior (2002) Lancet Neurol. 1,3).

Fragment Aβ15-25 and subfragments of 7-8 contiguous amino acids thereofare preferred because these peptides consistently generate a highimmunogenic response to Aβ peptide. These fragments include Aβ16-22,Aβ16-23, Aβ16-24, Aβ17-23, Aβ17-24, Aβ18-24, and Aβ18-25. Particularlypreferred Aβ15-25 subfragments are 7 contiguous amino acids in length.The designation Aβ15-21 for example, indicates a fragment includingresidues 15-21 of Aβ and lacking other residues of Aβ. and preferably7-10 contiguous amino acids. These fragments can generate an antibodyresponse that includes end-specific antibodies.

Peptide immunogens of Aβs require screening for activity in clearing orpreventing amyloid deposits (see WO 00/72880, which is incorporatedherein in its entirety for all purposes). Administration of N-terminalfragments of Aβ induces the production of antibodies that recognize Aβdeposits in vivo and in vitro. Fragments lacking at least one, andsometimes at least 5 or 10 C-terminal amino acids present in naturallyoccurring forms of Aβ are used in some methods. For example, a fragmentlacking 5 amino acids from the C-terminal end of Aβ43 includes the first38 amino acids from the N-terminal end of Aβ.

Unless otherwise indicated, reference to Aβ includes the natural humanamino acid sequences indicated above as well as analogs includingallelic, species and induced variants. Analogs typically differ fromnaturally occurring peptides at one, two or a few positions, often byvirtue of conservative substitutions. Analogs typically exhibit at least80 or 90% sequence identity with natural peptides. Some analogs alsoinclude unnatural amino acids or modifications of N- or C-terminal aminoacids at one, two, or a few positions. For example, the natural asparticacid residue at position 1 and/or 7 of Aβ can be replaced withiso-aspartic acid.

Examples of unnatural amino acids are D, alpha, alpha-disubstitutedamino acids, N-alkyl amino acids, lactic acid, 4-hydroxyproline,gamma-carboxyglutamate, epsilon-N,N,N-trimethyllysine,epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,omega-N-methylarginine, β-alanine, ornithine, norleucine, norvaline,hydroxproline, thyroxine, gamma-amino butyric acid, homoserine,citrulline, and isoaspartic acid. Immunogenic peptides also includeanalogs of Aβ and fragments thereof. Some therapeutic agents of theinvention are all-D peptides, e.g., all-D Aβ, all-D Aβ fragment, oranalogs of all-D Aβ or all-D Aβ fragment. Fragments and analogs can bescreened for prophylactic or therapeutic efficacy in transgenic animalmodels in comparison with untreated or placebo controls as described inWO 00/72880.

Peptide immunogens also include longer polypeptides that include, forexample, an immunogenic of Aβ peptide, together with other amino acids.For example, preferred immunogenic peptides include fusion proteinscomprising a segment of Aβ linked to a heterologous amino acid sequencethat induces a helper T-cell response against the heterologous aminoacid sequence and thereby a B-cell response against the Aβ segment. Suchpolypeptides can be screened for prophylactic or therapeutic efficacy inanimal models in comparison with untreated or placebo controls asdescribed in WO 00/72880.

The Aβ peptide, analog, immunogenic fragment or other polypeptide can beadministered in disaggregated or aggregated form. Disaggregated Aβ orfragments thereof means monomeric peptide units. Disaggregated Aβ orfragments thereof are generally soluble, and are capable ofself-aggregating to form soluble oligomers, protofibrils and ADDLs.Oligomers of Aβ and fragments thereof are usually soluble and existpredominantly as alpha-helices or random coils. Aggregated Aβ orfragments thereof means oligomers of Aβ or fragments thereof that haveassociate into insoluble beta-sheet assemblies. Aggregated Aβ orfragments thereof also means fibrillar polymers. Fibrils are usuallyinsoluble. Some antibodies bind either soluble Aβ or fragments thereofor aggregated Aβ or fragments thereof. Some antibodies bind both solubleAβ or fragments thereof and aggregated Aβ or fragments thereof.

Immunogenic peptides also include multimers of monomeric immunogenicpeptides. Immunogenic peptides other than Aβ peptides should induce animmunogenic response against one or more of the preferred fragments ofAβ listed above (e.g., Aβ1-3, 1-7, 1-10, and 3-7).

Immunogenic peptides of the present invention are linked to a carrierusing a method of the present invention to form a conjugate. Theimmunogenic peptide can be linked at its amino terminus, its carboxylterminus, or both to a carrier to form a conjugate. Optionally, multiplerepeats of the immunogenic peptide can be present in the conjugate.

An N-terminal fragment of Aβ can be linked at its C-terminus to acarrier peptide to form a conjugate. In such conjugates, the N-terminalresidue of the fragment of Aβ constitutes the N-terminal residue of theconjugate. Accordingly, such conjugates are effective in inducingantibodies that bind to an epitope that requires the N-terminal residueof Aβ to be in free form. Some immunogenic peptides of the inventioncomprise a plurality of repeats of an N-terminal segment of Aβ linked atthe C-terminus to one or more copy of a carrier peptide to form aconjugate. The N-terminal fragment of Aβ incorporated into suchconjugates sometimes begins at Aβ1-3 and ends at Aβ7-11. Aβ1-7, 1-3,1-4, 1-5, and 3-7 are preferred N-terminal fragment of Aβ. Someconjugates comprise different N-terminal segments of Aβ in tandem. Forexample, a conjugate can comprise Aβ1-7 followed by Aβ1-3 linked to acarrier.

In some conjugates, an N-terminal segment of Aβ is linked at itsN-terminal end to a carrier peptide. The same variety of N-terminalsegments of Aβ can be used as with C-terminal linkage. Some conjugatescomprise a carrier peptide linked to the N-terminus of an N-terminalsegment of Aβ, which is in turn linked to one or more additionalN-terminal segments of Aβ in tandem. Preferably, such immunogenic Aβfragments, once conjugated to an appropriate carrier, induce animmunogenic response that is specifically directed to the Aβ fragmentwithout being directed to other fragments of Aβ.

Immunogenic peptides of the invention include immunogenic heterologouspeptides. In some immunogenic peptides, an Aβ fragment is linked to acarrier to form an immunogenic heterologous peptide. This heterologouspeptide is linked to a carrier using a method of the present inventionto form a conjugate. Some of these immunogenic heterologous peptidescomprise fragments of Aβ linked to tetanus toxoid epitopes such asdescribed in U.S. Pat. No. 5,196,512, EP 378,881 and EP 427,347.Optionally, an immunogenic peptide can be linked to one or multiplecopies of a carrier, for example, at both the N and C termini of thecarrier to form an immunogenic heterologous peptide. Other of theseimmunogenic heterologous peptides comprise fragments of Aβ linked tocarrier peptides described in U.S. Pat. No. 5,736,142. For example, animmunogenic heterologous peptide can comprise Aβ1-7 followed by Aβ1-3followed by a carrier. Examples of such immunogenic heterologouspeptides include:

Aβ 1-7/Tetanus toxoid 830-844 + 947-967 in a linear configuration (SEQID NO.:24) DAEFRHD-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE

Peptides described in U.S. Pat. No. 5,736,142 (all in linearconfigurations):

PADRE/Aβ1-7: (SEQ ID NO.:26) AKXVAAWTLKAAA-DAEFRHD Aβ1-7 × 3/PADRE: (SEQID NO.:27) DAEFRHD-DAEFRHD-DAEFRHD-AKXVAAWTLKAAA PADRE/Aβ1-7 × 3: (SEQID NO.:28) AKXVAAWTLKAAA-DAEFRHD-DAEFRHD-DAEFRHD Aβ1-7/PADRE: (SEQ IDNO.:29) DAEFRHD-AKXVAAWTLKAAA Aβ1-7/albumin fragment: (SEQ ID NO.:30)DAEFRHD-ISQAVHAAHAEINEAGR Aβ4-10/albumin fragment: (SEQ ID NO.:31)FRHDSGY-ISQAVHAAHAEINLAGR Aβ3-9/albumin fragment: (SEQ ID NO.:32)EFRHDSG-ISQAVHAAHAEINEAGR HA₃₀₇₋₃₁₉/Aβ₁₋₇ × 3: (SEQ ID NO.:33)PKYVKQNTLKLAT-DAEFRHD-DAEFRHD-DAEFRHD Aβ₁₋₇/HA₃₀₇₋₃₁₉/Aβ₁₋₇: (SEQ IDNO.:34) DAEFRHD-PKYVKQNTLKLAT-DAEFRHD Aβ₁₋₇ × 3/HA₃₀₇₋₃₁₉: (SEQ IDNO.:35) DAEFRHD-DAEFRHD-DAEFRHD-PKYVKQNTLKLAT Aβ₁₋₇ × 2/HA₃₀₇₋₃₁₉: (SEQID NO.:36) DAEFRHD-DAEFRHD-PKYVKQNTLKLAT Aβ₁₋₇/HA₃₀₇₋₃₁₉/MalariaCS/TT₈₃₀₋₈₄₄/ TT₉₄₇₋₉₆₇/Aβ₁₋₇ (SEQ ID NO.:37)DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITEL-C-FNNFTVSF WLRVPKSASHLE Aβ₁₋₇× 3/TT₈₃₀₋₈₄₄/C/TT₉₄₇₋₉₆₇ (SEQ ID NO.:38)DAEFRHD-DAEFRHD-DAEFRHD-QYIKANSKFIGITEL-C-FNNFTVSF WLRVPKVSASHLEAβ₁₋₇/TT₈₃₀₋₈₄₄/C/TT₉₄₇₋₉₆₇ (SEQ ID NO.:39)DAEFRHD-QYIKANTSKFIGITELCFNNFTVSFWLRVPKVSASHLEAβ₁₋₇/TT₈₃₀₋₈₄₄/C/TT₉₄₇₋₉₆₇/Aβ₁₋₇ (SEQ ID NO.:20)DAEFRHD-QYIKANSKFIGITEL-C-FNNFTVSFWLRVPKVSASHLE-DP FPHD

Some immunogenic heterologous peptides comprise a multimer ofimmunogenic peptides represented by the formula 2^(x), in which x is aninteger from 1-5. Preferably x is 1, 2 or 3, with 2 being mostpreferred. When x is two, such a multimer has four immunogenic peptideslinked in a preferred configuration referred to as MAP4 (see U.S. Pat.No. 5,229,490). Such immunogenic peptides are then linked to a carrierusing a method of the present invention to form a conjugate.

The MAP4 configuration is shown below, where branched structures areproduced by initiating peptide synthesis at both the N-terminal and sidechain amines of lysine. Depending upon the number of times lysine isincorporated into the sequence and allowed to branch, the resultingstructure will present multiple N-termini. In this example, fouridentical N-termini have been produced on the branched lysine-containingcore. Such multiplicity greatly enhances the responsiveness of cognate Bcells.

Examples of such immunogenic heterologous peptides include:

Aβ 1-7/Tetanus toxoid 830-844 in a MAP4 configuration: (SEQ ID NO.:22)DAEFRHD-QYIKANSKFIGITEL Aβ 1-7/Tetanus toxoid 947-967 in a MAP4configuration: (SEQ ID NO.:23) DAEFRHD-FNNFTVSFWLRVPKVSASHLEAβ 3-9/Tetanus toxoid 830-844 in a MAP4 configuration: (SEQ ID NO.:25)EFRHDSG-QYIKANSKFIGITELDAEFRHD-QYIKANSKFIGITEL on a 2 branched resin

The Aβ peptide, analog, active fragment or other polypeptide can beadministered in associated or multimeric form or in dissociated form.Therapeutic agents also include multimers of monomeric immunogenicagents. Agents other than Aβ peptides should induce an immunogenicresponse against one or more of the preferred fragments of Aβ listedabove (e.g., 1-10, 1-7, 1-3, and 3-7), and can also be conjugated to acarrier using a method of the present invention. Preferably, suchagents, once conjugated to an appropriate carrier, induce an immunogenicresponse that is specifically directed to one of these fragments withoutbeing directed to other fragments of Aβ. To facilitate the conjugationof an peptide immunogen with a carrier, additional amino acids can beadded to the termini of the antigenic determinants. The additionalresidues can also be used for modifying the physical or chemicalproperties of the peptide immunogen. Amino acids such as tyrosine,cysteine, lysine, glutamic or aspartic acid, or the like, can beintroduced at the C- or N-terminus of the peptide immunogen.Additionally, peptide linkers containing amino acids such as glycine andalanine can also be introduced. In addition, the antigenic determinantscan differ from the natural sequence by being modified by terminalNH₂-group acylation, e.g., by alkanoyl (C1-C20) or thioglycolylacetylation, terminal-carboxy amidation, e.g., ammonia, methylamine,etc. In some instances these modifications may provide sites for linkingto a support or other molecule.

The peptide immunogens used to generate conjugates of the presentinvention using a process disclosed herein can be combined via linkageto form polymers (multimers), or can be formulated in a compositionwithout linkage, as an admixture. Where a peptide is linked to anidentical peptide, thereby forming a homopolymer, a plurality ofrepeating epitopic units are presented. For example, multiple antigenpeptide (MAP) technology is used to construct polymers containing bothCTL and/or antibody peptides and peptides. A “CTL epitope” is onederived from selected eptiopic regions of potential target antigens.When the peptides differ, e.g., a cocktail representing different viralsubtypes, different epitopes within a subtype, different HLA restrictionspecificities, or peptides which contain T-helper epitopes,heteropolymers with repeating units are provided. In addition tocovalent linkages, noncovalent linkages capable of formingintermolecular and intrastructural bonds are also contemplated.

Such peptide immunogens and their analogs are synthesized by solid phasepeptide synthesis or recombinant expression, or are obtained fromnatural sources. Automatic peptide synthesizers are commerciallyavailable from numerous suppliers, such as Applied Biosystems, FosterCity, Calif.

Recombinant expression can be in bacteria (such as E. coli), yeast,insect cells or mammalian cells. Procedures for recombinant expressionare described by Sambrook et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Press, NY, 2nd ed., 1989). Some immunogenic peptidesare also available commercially (e.g., American Peptides Company, Inc.,Sunnyvale, Calif., and California Peptide Research, Inc., Napa, Calif.).

Random libraries of peptides or other compounds can also be screened forsuitability as a peptide immunogen. Combinatorial libraries can beproduced for many types of compounds that can be synthesized in astep-by-step fashion. Such compounds include polypeptides, beta-turnmimetics, hormones, oligomeric N-substituted glycines, andoligocarbamates and the like. Large combinatorial libraries of thecompounds can be constructed by the encoded synthetic libraries (ESL)method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503and WO 95/30642 (each of which is incorporated by reference for allpurposes). Peptide libraries can also be generated by phage displaymethods (see, e.g., Devlin, WO 91/18980).

Derivatization and Conjugation of an Immunogenic Peptide to a ProteinCarrier

The site of attachment of a peptide immunogen to a protein/polypeptidecarrier, and the nature of the cross-linking agent that is used toattach a peptide immunogen to the carrier are both important to thespecificity of the resultant antibody generated against it. For properrecognition, the peptide immunogen must be coupled to the carrier withthe appropriate orientation. For an antibody to recognize subsequentlythe free peptide immunogens without carrier, the peptideimmunogen-protein/polypeptide carrier conjugate must present the peptideimmunogens in an exposed and accessible form. Optimal orientation isoften achieved by directing the cross-linking reaction to specific siteson the peptide immunogens. One way to achieve this with a peptideimmunogen is by attaching a terminal cysteine residue during peptidesynthesis. This provides a sulfhydryl group on one end of the peptidefor conjugation to the carrier. Cross-linking through this groupprovides attachment of the peptide immunogen only at one end, therebyensuring consistent orientation.

In peptide immunogen-carrier conjugation, the goal is not to maintainthe native state or stability of the carrier, but to present the haptenin the best possible way to the immune system. In reaching this goal,the choice of conjugation chemistry may control the resultant titer,affinity, and specificity of the antibodies generated against thehapten. It may be important in some cases to choose a cross-linkingagent containing a spacer arm long enough to present the antigen in anunrestricted fashion. It also may be important to control the density ofthe peptide immunogen on the surface of the carrier. Too little peptideimmunogen substitution may result in little or no response. A peptideimmunogen density too high actually may cause immunological suppressionand decrease the response. In addition, the cross-linker itself maygenerate an undesired immune response. These issues need to be takeninto consideration in selecting not only the appropriate cross-linkingreagents, but also the appropriate ratios of protein/polypeptide carrierand peptide immunogen.

A variety of means of attaching the protein/peptide carriers to thepeptide immunogens are possible. Ionic interactions are possible throughthe termini or through the ε-amino group of lysine. Hydrogen bondingbetween the side groups of the residues and the peptide immunogen arealso possible. Finally, conformation interactions between theprotein/peptide carriers and the immunogenic peptide may give rise to astable attachment.

Peptide immunogens-carrier conjugates have been successfully generatedusing various cross-linking reagents such as zero-length,homobifunctional or heterobifunctional cross linkers. The smallestavailable reagent systems for bioconjugation are the so-calledzero-length cross-linkers. These compounds mediate the conjugation oftwo molecules by forming a bond containing no additional atoms. Thus,one atom of a molecule is spacer. In many conjugation schemes, the finalcomplex is bound together by virtue of chemical components that addforeign structures to the substances being cross-linked. In someapplications, the presence of these intervening linkers may bedetrimental to the intended use. For instance, in the preparation ofpeptide immunogen-carrier conjugates the complex is formed with theintention of generating an immune response to the attached hapten.Occasionally, a portion of the antibodies produced by this response willhave specificity for the cross-linking agent used in the conjugationprocedure. Zero-length cross-linking agents eliminate the potential forthis type of cross-reactivity by mediating a direct linkage between twosubstances.

Homobifunctional reagents, which were the first cross-linking reagentsused for modification and conjugation of macromolecules, consisted ofbireactive compounds containing the same functional group at both ends(Hartman and Wold, 1966). These reagents could tie one protein toanother by covalently reacting with the same common groups on bothmolecules. Thus, the lysine F-amines or N-terminal amines of one proteincould be cross-linked to the same functional groups on a second proteinsimply by mixing the two together in the presence of thehomobifunctional reagent.

Heterobifunctional conjugation reagents contain two different reactivegroups that can couple to two different functional targets on proteinsand other macromolecules. For example, one part of a cross-linker maycontain an amine-reactive group, while another portion may consist of asulfhydryl-reactive group. The result is the ability to direct thecross-linking reaction to selected parts of target molecules, thusgarnering better control over the conjugation process.

Heterobifunctional reagents are used to cross-link proteins and othermolecules in a two- or three-step process that limits the degree ofpolymerization often obtained using homobifunctional cross-linkers.

Many methods are currently available for coupling of peptide immunogensto protein/polypeptide carriers using zero-length, homobifunctional orheterobifunctional crosslinkers. Most methods create amine, amide,urethane, isothiourea, or disulfide bonds, or in some cases thioethers.The more general method of coupling proteins or peptides to peptidesutilizes bifunctional crosslinking reagents. These are small spacermolecules having active groups at each end. The spacer molecules canhave identical or different active groups at each end. The most commonactive functionalities, coupling groups, and bonds formed are:

-   -   1. Aldehyde—amino→secondary amine    -   2. Maleimido—sulfhydryl (thioether    -   3. Succinimido—amino (amide    -   4. Imidate esters—amino (−amide    -   5. Phenyl azides—amino (phenyl amine    -   6. Acyl halide—sulfhydryl (thioether    -   7. Pyridyldisulfides—sulfhydryl (disulfide    -   8. Isothiocyanate—amino (isothiourea.

The reactivity of a given carrier protein, in terms of its ability to bemodified by a cross-linking agent such that it can be conjugated to anpeptide immunogen, is determined by its amino acid composition and thesequence location of the individual amino acids in the three dimensionalstructure of the molecule, as well as by the amino acid composition ofthe peptide immunogen.

In the case of linkers (“L”) between protein/peptide carriers and otherpeptides (e.g., a protein/peptide carriers and an peptide immunogen),the spacers are typically selected from Ala, Gly, or other neutralspacers of nonpolar amino acids or neutral polar amino acids. In certainembodiments the neutral spacer is Ala. It will be understood that theoptionally present spacer need not be comprised of the same residues andthus may be a hetero- or homo-oligomer. Exemplary spacers includehomo-oligomers of Ala. When present, the spacer will usually be at leastone or two residues, more usually three to six residues. In otherembodiments the protein/polypeptide carrier is conjugated to an peptideimmunogen, preferably with the protein/peptide carrier positioned at theamino terminus. The peptide may be joined by a neutral linker, such asAla-Ala-Ala or the like, and preferably further contain a lipid residuesuch palmitic acid or the like which is attached to alpha and epsilonamino groups of a Lys residue ((PAM)2Lys), which is attached to theamino terminus of the peptide conjugate, typically via Ser-Ser linkageor the like.

In some aspects of the invention, the peptide immunogen is an Aβfragment selected from the group consisting of Aβ1-5-L, Aβ1-7-L,Aβ1-9-L, and Aβ1-12-L. In some aspects of the invention the linker isGAGA (SEQ ID NO:10).

To facilitate the conjugation of a peptide immunogen with a carrier,additional amino acids can be added to the termini of the antigenicdeterminants. The additional residues can also be used for modifying thephysical or chemical properties of the peptide immunogen. Amino acidssuch as tyrosine, cysteine, lysine, glutamic or aspartic acid, or thelike, can be introduced at the C- or N-terminus of the peptideimmunogen. Additionally, peptide linkers containing amino acids such asglycine and alanine can also be introduced. In addition, the antigenicdeterminants can differ from the natural sequence by being modified byterminal NH₂-group acylation, e.g., by alkanoyl (C1-C20) or thioglycolylacetylation, terminal-carboxy amidation, e.g., ammonia, methylamine,etc. In some instances these modifications may provide sites for linkingto a support or other molecule.

In some aspects of the invention, the peptide immunogen is an Aβfragment selected from the group consisting of Aβ1-5-C, Aβ1-7-C,Aβ1-9-C, and Aβ1-12-C, where C is a cysteine amino acid residue. In someaspects of the invention, the peptide immunogen is an Aβ fragmentselected from the group consisting of Aβ1-5-L-C, Aβ1-7-L-C, Aβ1-9-L-C,and Aβ1-12-L-C.

The peptide immunogen is linked to the protein/peptide carrier eitherdirectly or via a linker either at the amino or carboxy terminus of thepeptide immunogen. The amino terminus of either the peptide immunogen orthe protein/peptide carrier may be acylated. In addition, the peptideimmunogen-protein/peptide carrier conjugate may be linked to certainalkanyol (C₁-C₂₀) lipids via one or more linking residues such as Gly,Gly-Gly, Ser, Ser-Ser as described below. Other useful lipid moietiesinclude cholesterol, fatty acids, and the like.

Peptide immunogens can be linked to a carrier by chemical crosslinking.Techniques for linking an immunogen to a carrier include the formationof disulfide linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate(SPDP) (Carlsson, J et al. (1978) Biochem J, 173: 723,) and succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptidelacks a sulfhydryl group, this can be provided by addition of a cysteineresidue to the hapten). These reagents create a disulfide linkagebetween themselves and peptide cysteine resides on one protein and anamide linkage through the s-amino on a lysine, or other free amino groupin other amino acids. A variety of such disulfide/amide-forming agentsare described in Immuno. Rev. 62: 85 (1982). Other bifunctional couplingagents form a thioether rather than a disulfide linkage. The thioetherforming agents include reactive ester of 6-maleimidocaproic acid,2-bromoacetic acid, and 2-iodoacetic acid,4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl groupscan be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.

Most frequently, lysine residues are the most abundant amino acidresidues found on carrier proteins, and these residues are modifiedusing cross-linking reagents to generate nucleophilic sites that arethen coupled to a hapten. This coupling is achieved via any of thehydrophilic side chains on the hapten molecules that are chemicallyactive. These include the guanidyl group of arginine, the (-carboxylgroups of glutamate and aspartic acid, the sulfhydryl group of cysteine,and the ε-amino group of lysine, to name a few. Modification of proteinssuch that they can now be coupled to other moieties is achieved usingcrosslinking reagents, which react with any of the side chains on theprotein carrier or hapten molecules.

In one aspect of the present invention, the carrier protein with orwithout a linker molecule is functionalized (derivatized) with a reagentthat introduces reactive sites into the carrier protein molecule thatare amenable to further modification to introduce nucleophilic groups.In one embodiment, the carrier is reacted with a haloacetylatingreagent, which preferentially reacts with a number of functional groupson amino acid residues of proteins such as the sulfhydryl group ofcysteine, the primary ε-amine group of lysine residue, the α terminal ofα-amines, the thioether of methionine and both imidazoyl side chainnitrogens of histidine (Gurd, 1967). In a preferred embodiment, theprimary ε-amine groups on lysine residues of the carrier protein arederivatized with b N-hydroxysuccinimidyl bromoacetate to generate abromoacetylated carrier. Conjugation of peptide immunogen and theactivated protein carrier was carried out by slowly adding the activatedcarrier to the solution containing the peptide immunogen.

By using the process of this invention, the peptide immunogens discussedin section B, above, may be conjugated to any of the carriers discussedin section A, above. The conjugates resulting from the process of thisinvention are used as immunogens for the generation of antibodiesagainst Aβ for use in passive/active immunotherapy. Furthermore, Aβ oran Aβ fragment linked to a carrier can be administered to a laboratoryanimal in the production of monoclonal antibodies to Aβ.

In one aspect of the invention, the conjugate is a conjugate selectedfrom the group consisting of Aβ1-7-CPM₁₉₇, (Aβ1-7×3)-CRM₁₉₇, and(Aβ1-7×5)-CRM₁₉₇. In one aspect of the invention, the conjugate is aconjugate selected from the group consisting of CRM₁₉₇-Aβ1-5,CRM₁₉₇-Aβ1-7, CRM₁₉₇-Aβ1-9, and CRM₁₉₇-Aβ1-12. In another aspect of theinvention, the conjugate is a conjugate selected from the groupconsisting of Aβ1-5-C-CRM₁₉₇, Aβ1-7-C-CRM₁₉₇, Aβ1-9-C-CRM₁₉₇, andAβ1-12-C-CRM₁₉₇, Aβ16-23-C-CRM₁₉₇, Aβ17-24-C-CRM₁₉₇, Aβ18-25-C-CRM₁₉₇,CRM₁₉₇-C-Aβ16-23, CRM₁₉₇-C-Aβ17-24, CRM₁₉₇-C-Aβ18-25, Aβ16-22-C-CRM₁₉₇,Aβ17-23-C-CRM₁₉₇, A18-24-C-CRM₁₉₇, CRM₁₉₇-C-Aβ16-22, CRM₁₉₇-C-Aβ17-23,and CRM₁₉₇-C-Aβ18-24. Aβ1-9-C-CRM₁₉₇, and Aβ1-12-C-CRM₁₉₇. In yetanother aspect of the invention, the conjugate is a conjugate selectedfrom the group consisting of selected from the group consisting ofAβ1-5-L-C-CRM₁₉₇, Aβ1-7-L-C-CRM₁₉₇, Aβ1-9-L-C-CRM₁₉₇, andAβ1-12-L-C-CRM₁₉₇.

Capping

A disadvantage to the use of coupling reagents, which introduce reactivesites into the side chains of reactive amino acid molecules on carrierand/or hapten molecules, is that the reactive sites if not neutralizedare free to react with any unwanted molecule either in vitro or in vivo.In the process of the present invention, capping of the unreactedfunctional groups is accomplished by reaction of the conjugates withpendant reactive groups with reagents which inactivate/cap the reactivegroups. Exemplary inactivating/capping reagents for use with theconjugation process of the present invention include cysteamine,N-acetylcysteamine, and ethanolamine. Alternatively, capping isaccomplished by reaction with ammonia or ammonium bicarbonate, either ofwhich converts the haloacetyl groups to aminoacetyl groups. Capping isalso accomplished at alkaline pH (9.0-9.8) using sodium hydroxide orsodium carbonate, which converts the haloacetyl groups to hydroxyacetylgroups. One potential advantage of converting the haloacetyl groups toaminoacetyl or hydroxyacetyl groups, as opposed to the reaction withcysteamine derivatives, ethanolamine etc., is the introduction ofrelatively smaller size chemical functionalities, by reaction withammonia or hydroxide/carbonate. The resulting capped functional groups,e.g. aminoacetyl or hydroxyacetyl, provide relatively less perturbancein the carrier protein portion of the conjugate. The capped peptideimmunogen-carrier protein is purified as necessary using known methods,such as chromatography (gel filtration, ion exchange, hydrophobicinteraction or affinity), dialysis, ultrafiltration-diafiltration,selective precipitation using ammonium sulfate or alcohol, and the like.

Immunogenic Conjugates and Compositions

The capped peptide immunogen-carrier protein conjugates are administeredin an immunogenic composition to mammals, particularly humans, forprophylactic and/or therapeutic purposes. The conjugates of the presentinvention are used to elicit and/or enhance immune responses againstimmunogens. For instance, CTL-carrier conjugates are used to treatand/or prevent viral infection, amyloidogenic diseases, cancer etc.Alternatively, polypeptide immunogen-carrier conjugates, which induceantibody responses, are also used.

In therapeutic applications, a conjugate of the present invention isadministered to an individual already suffering from an amyloidogenicdisease such as Alzheimer's disease. Those in the incubation phase orthe acute phase of the disease may be treated with the conjugate of thepresent invention separately or in conjunction with other treatments, asappropriate.

In therapeutic applications, an immunogenic composition of the presentinvention is administered to a patient in an amount sufficient to elicitan effective CTL response or humoral response to the amyloid plaque, andto cure, or at least partially arrest disease progression, symptomsand/or complications. An amount adequate to accomplish this is definedas “therapeutically effective dose.” Amounts effective for this use willdepend in part on the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician.

Therapeutically effective amounts of the immunogenic compositions of thepresent invention generally range for the initial immunization fortherapeutic or prophylactic administration, from about 0.1 μg to about10,000 μg of peptide for a 70 kg patient, usually from about 0.1 toabout 8000 μg, preferably between about 0.1 to about 5000 μg, and mostpreferably between 0.1 to about 1,000 μg. These doses are followed byboosting dosages of from about 0.1 μg to about 1000 μg of peptidepursuant to a boosting regimen over weeks to months depending upon thepatient's response and condition by measuring specific immune responses.

Further, the present invention is used prophylactically to preventand/or ameliorate amyloidogenic disease. Effective amounts are asdescribed above. Additionally, one of ordinary skill in the art wouldalso know how to adjust or modify prophylactic treatments, asappropriate, for example by boosting and adjusting dosages and dosingregimes.

Therapeutic administration may begin at the first sign of the disease.This is followed by boosting doses until the disease progression ishalted or reversed or the symptoms are substantially abated and for aperiod thereafter.

Immunogenic compositions of the present invention for therapeutic orprophylactic treatment can be administered by parenteral, topical,intravenous, oral, subcutaneous, intra-arterial, intra-cranial,intra-peritoneal, intra-nasal or intra-muscular means for prophylacticand/or therapeutic treatment. One typical route of administration of animmunogenic agent is subcutaneous, although other routes can be equallyeffective. Another common route is intra-muscular injection. This typeof injection is most typically performed in the arm or leg muscles. Insome methods, agents are injected directly into a particular tissuewhere deposits have accumulated, for example intra-cranial injection.Intra-muscular injection or intravenous infusion is preferred foradministration of antibody. In some methods, particular therapeuticantibodies are injected directly into the cranium. Because of the easeof administration, the immunogenic compositions of the invention areparticularly suitable for oral administration. The invention furtherprovides immunogenic compositions for parenteral administration, whichcomprise a solution of the peptides or conjugates, dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier.

A variety of diluents, excipients and buffers may be used, e.g., water,buffered water, phosphate buffered saline, 0.3% glycine, hyaluronic acidand the like. These compositions may be sterilized by conventional,well-known sterilization techniques, or may be sterile filtered. Theresulting aqueous solutions may be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterilesolution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may beused. These may include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more conjugates of the invention, and morepreferably at a concentration of 25-75%.

The concentration of immunogenic compositions of the present inventionin the pharmaceutical formulations can vary widely, i.e., from less thanabout 0.1%, usually at or at least about 2% to as much as 20% to 50% ormore by weight, and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected.

The conjugates of the present invention may also be administered vialiposomes, which serve to target the conjugates to a particular tissue,such as lymphoid tissue, or targeted selectively to infected cells, aswell as increase the half-life of the peptide composition. Liposomesinclude emulsions, foams, micelles, insoluble monolayers, liquidcrystals, phospholipid dispersions, lamellar layers and the like. Inthese preparations the composition to be delivered is incorporated aspart of a liposome, alone or in conjunction with a molecule, which bindsto, for example, a receptor prevalent among lymphoid cells. Thesemolecules would include monoclonal antibodies, which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes filled with a desired composition of the present invention canbe directed to the site of lymphoid cells, where the liposomes thendeliver the selected therapeutic/immunogenic peptide compositions.Liposomes for use in the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos.4,235,871, 4,501,728, 4,837,028, and 5,019,369, incorporated herein byreference.

For aerosol administration, the compositions of the present inventionare preferably supplied in finely divided form along with a surfactantand propellant. Typical percentages of the composition are 0.01-20% byweight, preferably 1-10%. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of such agentsare the esters or partial esters of fatty acids containing from 6 to 22carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic,linoleic, linolenic, olesteric and oleic acids with an aliphaticpolyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixedor natural glycerides may be employed. The surfactant may constitute0.1-20% by weight of the composition, preferably 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, if desired, as with lecithin for intranasal delivery.

Conjugates of the present invention can optionally be administered incombination with other agents that are at least partly effective intreatment and/or amelioration of a an amyloid disease and/or itssymptoms. In the case of Alzheimer's and Down's syndrome, in whichamyloid deposits occur in the brain, the conjugates of the invention canbe administered in conjunction with other agents that increase passageof the agents of the invention across the blood-brain barrier.

The immunogenic composition typically contains an adjuvant. An adjuvantis a substance that enhances the immune response when administeredtogether with an immunogen or antigen. A number of cytokines orlymphokines have been shown to have immune modulating activity, and thusmay be used as adjuvants, including, but not limited to, theinterleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, e.g., U.S. Pat.No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms), theinterferons-α, β and γ, granulocyte-macrophage colony stimulating factor(see, e.g., U.S. Pat. No. 5,078,996) macrophage colony stimulatingfactor, granulocyte colony stimulating factor, GSF, and the tumornecrosis factor α and β. Still other adjuvants useful in this inventioninclude a chemokine, including without limitation, MCP-1, MIP-1α,MIP-1β, and RANTES. Adhesion molecules, such as a selectin, e.g.,L-selectin, P-selectin and E-selectin may also be useful as adjuvants.Still other useful adjuvants include, without limitation, a mucin-likemolecule, e.g., CD34, GlyCAM-1 and MadCAM-1, a member of the integrinfamily such as LFA-1, VLA-1, Mac-1 and p150.95, a member of theimmunoglobulin super family such as PECAM, ICAMs, e.g., ICAM-1, ICAM-2and ICAM-3, CD2 and LFA-3, co-stimulatory molecules such as CD40 andCD40L, growth factors including vascular growth factor, nerve growthfactor, fibroblast growth factor, epidermal growth factor, B7.2, PDGF,BL-1, and vascular endothelial growth factor, receptor moleculesincluding Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3,AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Stillanother adjuvant molecule includes Caspase (ICE). See, alsoInternational Patent Publication Nos. WO98/17799 and WO99/43839, whichare incorporated herein by reference in their entirety for all purposes.

Suitable adjuvants used to enhance an immune response include, withoutlimitation, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa,Hamilton, Mont.), which is described in U.S. Pat. No. 4,912,094, whichis hereby incorporated by reference for all purposes. Also suitable foruse as adjuvants are synthetic lipid A analogs or aminoalkyl glucosaminephosphate compounds (AGP), or derivatives or analogs thereof, which areavailable from Corixa (Hamilton, Mont.), and which are described in U.S.Pat. No. 6,113,918, which is hereby incorporated by reference. One suchAGP is 2-[(R)-3-Tetradecanoyloxytetradecancylamino]ethyl2-Deoxy-4-O-phosphono-3-O—[(S)-3-tetradecanoyoxytetradecanoyl]-2-[(R)-3-tetradecanoyloxy-tetradecanoyl-amino]-b-D-glycopyranoside,which is known as 529 (also known as RC529; Corixa). This 529 adjuvantis formulated as an aqueous form (529 AF) or as a stable emulsion (529SE).

Still other adjuvants include mineral oil and water emulsions, calciumsalts such as calcium phosphate, aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, etc., Amphigen, Avridine, L121/squalene,D-lactide-polylactide/glycoside, pluronic acids, polyols, muramyldipeptide, killed Bordetella, saponins, such as Stimulon™ QS-21(Antigenics, Framingham, Mass.), described in U.S. Pat. No. 5,057,540,which is hereby incorporated by reference3, and particles generatedtherefrom such as ISCOMS (immunostimulating complexes), Mycobacteriumtuberculosis, bacterial lipopolysaccharides, synthetic polynucleotidessuch as oligonucleotides containing a CpG motif (U.S. Pat. No.6,207,646, which is hereby incorporated by reference), a pertussis toxin(PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72,PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302and WO 92/19265, which are/incorporated herein by reference for allpurposes.

Also useful as adjuvants are cholera toxins and mutants thereof,including those described in published International Patent ApplicationNo. WO 00/18434 (wherein the glutamic acid at amino acid position 29 isreplaced by another amino acid (other than aspartic acid, preferably ahistidine). Similar CT toxins or mutants are described in publishedInternational Patent Application number WO 02/098368 (wherein theisoleucine at amino acid position 16 is replaced by another amino acid,either alone or in combination with the replacement of the serine atamino acid position 68 by another amino acid; and/or wherein the valineat amino acid position 72 is replaced by another amino acid). Other CTtoxins are described in published International Patent Applicationnumber WO 02/098369 (wherein the arginine at amino acid position 25 isreplaced by another amino acid; and/or an amino acid is inserted atamino acid position 49; and/or two amino acids are inserted at aminoacid position 35 and 36).

It is to be understood that reference throughout this specification toany theory to explain the results described is not to limit the scope ofthe invention. Independent of the method by which the inventionfunctions, the results and advantages described herein may be achievedby reference to the following examples of the invention.

It will be apparent to one of ordinary skill in the art that manychanges and modifications can be made thereto without departing from thespirit or scope of the appended claims. All publications, patents andpatent applications mentioned in this specification are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by reference.

EXAMPLE 1 Conjugation of CRM₁₉₇ to Aβ Peptide

Conjugation of haptens/antigenic peptides was carried out by reactingactivated carrier CRM₁₉₇, which has thirty-nine lysine residues, to ahapten/antigenic peptide having a pendant thiol-group using the methoddescribed below (FIG. 1). All the Aβ peptides contained a cysteineresidue at the carboxy terminus to facilitate the conjugation of thesepeptides through the cysteinyl sulfhydryl group to the carrier protein.These peptides were produced by solid phase synthesis.

I. Activation

Free amino groups of CRM₁₉₇ were bromoacetylated by reaction with anexcess of bromoacetic acid N-hydroxysuccinimide ester (Sigma ChemicalCo., St. Louis, Mo.) (Bernatowicz and Matsueda, 1986). To an ice-coldsolution of CRM₁₉₇ (˜15 mg), 10% (v/v) 1.0 M NaHCO₃ (pH 8.4) was added.Bromoacetic acid N-hydroxysuccinimide ester, equal in weight to that ofCRM₁₉₇ used, was dissolved in 200 μL dimethylformamide (DMF), addedslowly to the CRM₁₉₇, and gently mixed at room temperature in the darkfor 1 hour. The resulting bromoacetylated (activated) protein waspurified by passage through a desalting (P6-DG) column using PBS/1 mMEDTA (pH 7.0) as the eluent. Following purification, the fractionscorresponding to activated CRM₁₉₇ were pooled and the proteinconcentration was estimated by BCA protein assay. The protein aminogroups, both before and after treatment with bromoacetic acidN-hydroxysuccinimide ester, were reacted with2,4,6-trinitrobenzenesulfonic acid (TNBSA), which served as an indicatorof bromoacetylation (Means et al., 1972).

II. Conjugation

Prior to conjugation, the peptides were reacted with5,5′-dithio-bis(2-nitrobenzoic acid) [Ellman's reagent] to verify thecontent of free —SH groups (between 62-88% reduced). For the first fourAβ peptides (amino acids 1-7 without linker, amino acids 1-12 with GAGA(SEQ ID NO.:10) linker, amino acids 1-9 with GAGA (SEQ ID NO.: 10)linker, and amino acids 1-7 with GAGA (SEQ ID NO.: 10) linker),approximately 8.0-10.0 mg of peptide was dissolved in sterile distilledwater to an approximate concentration of 20 mg/ml. The peptide wasslowly added to cold activated CRM₁₉₇ in a 1:1 ratio (w/w) and the pHwas adjusted to approximately 7.0-7.2 with the addition of 20-36 ft of 1N NaOH. The resulting material was gently mixed overnight at 4° C. inthe dark followed by dialysis in the dark against two 1 L changes ofPBS, pH 7.2. For the next four Aβ peptides (amino acids 1-5 withoutlinker, amino acids 1-9 without linker, amino acids 1-12 without linker,and amino acids 1-5 with linker), reaction with Ellman's reagent wasused to verify the free —SH groups. CRM₁₉₇ was bromoacetylated,purified, and reacted with TNBSA as previously described. The pH of eachpeptide was adjusted to 7.0 with the addition of 0.1 M NaPO₄ (pH 8.5) at2.2× the volume of the dissolved peptide. The peptide was slowly addedto cold activated CRM₁₉₇ in a 1:1 ratio and allowed to react overnightat 4° C. in the dark. The resulting material was dialyzed. A finalcontrol peptide (1-12mer in reverse orientation) was conjugated toCRM₁₉₇ as described above with the following modification. Rather thanadjusting the pH of the peptide to 7.0, the pH of the activated CRM₁₉₇was adjusted to approximately 7.5 with the addition of 20% (v/v) 0.5 MNaPO₄ (pH 8.0). Each conjugate, after dialysis, was transferred into asterile 15 mL polypropylene tube, wrapped in aluminum foil, and storedat 4° C. Activation of the reactive amino residues on the carrier wasthen subsequently verified using mass spectrometry.

Conjugate Immunogenic Peptide A□1-5-C-CRM₁₉₇ DAEFR-C (SEQ. ID. NO.:1)A□1-7-C-CRM₁₉₇ DAEFRHD-C (SEQ. ID NO.:2) Aβ1-9-C-CRM₁₉₇ DAEFRHDSG-C (SEQID NO:3) Aβ1-12-C-CRM₁₉₇ DAEFRHDSGYEV-C (SEQ ID NO:4) Aβ1-5-L-C-CRM₁₉₇DAEFR-GAGA-C (SEQ ID NO.:5) Aβ1-7-L-C-CRM₁₉₇ DAEFRHD-GAGA-C (SEQ IDNO.:6) Aβ1-9-L-C-CRM₁₉₇ DAEFRHDSG-GAGA-C (SEQ ID NO.:7)Aβ1-12-L-C-CRM₁₉₇ DAEFRHDSGYEV-GAGA-C (SEQ ID NO.:8)Aβ2-1-C-CRM_(197 (−VE CONTROL)) VEYGSDHRFEAD-C (SEQ ID NO.:9) L = linker(GAGA) (SEQ ID NO.:10)

EXAMPLE 2 Preparation of Aβ Peptide-CRM₁₉₇ Conjugate and Purification byUltrafiltration Bromoacetylation of CRM₁₉₇

CRM₁₉₇ (100 mg) in 0.01 M sodium phosphate buffer, 0.9% NaCl, pH 7.0,was reacted with bromoacetic acid N-hydroxysucinimide ester (dissolvedto 20 mg/mL in DMSO) at a 1:1 weight ratio under an argon atmosphere.The reaction was titrated as needed to maintain the pH at 7.0. Themixture was stirred in dark for 1.5 hours at room temperature. Thereaction mixture was 1.2 μm filtered into the retentate reservoir of aUF/DF system (Millipore Labscale TFF, Billerica, Mass.). Purificationwas done using a 10K or 30K UF membrane by diafiltration (30-fold)against 0.01 M sodium phosphate buffer/0.9% NaCl, pH 7.0. Thebromoacetylated CRM₁₉₇ was filtered by passing through a 0.2 μm filter.The degree of bromoacetylation was determined by reacting the activatedCRM₁₉₇ with cysteine, followed by amino acid analysis and quantitationof the resulting carboxymethylcysteine (CMC).

Conjugation of Aβ Peptide and Bromoacetylated CRM₁₉₇ and Capping withN-Acetylacysteamine

Bromoacetylated CRM₁₉₇ (50 mg) was transferred to a reaction vessel. Tothe stirred solution, maintained at 2-8° C., was added 1 M sodiumcarbonate/bicarbonate. Titration was performed to achieve a target pH of9.0, under argon atmosphere. Separately, 50 mg of Aβ peptide was weighedout and dissolved in water for injection (WFI) to 20 mg/mL. To thissolution was added 1 M sodium carbonate/bicarbonate until pH 9.0 wasattained. The peptide solution was added to the bromoacetylated CRM₁₉₇solution, and the mixture was stirred at 2-8° C. for 14-18 hours. Theremaining bromoacetyl groups were capped with a 20-fold molar excess ofN-acetylcysteamine for 3-6 hours at 2-8° C.

The reaction mixture was filtered through 1.2 μm filter into theretentate reservoir of a UF/DF system (Millipore XL), and the conjugatewas purified at room temperature by 30-fold diafiltration on a 10K or30K MWCO membrane (Millipore) by diafiltering against 0.01 M sodiumphosphate buffer/0.9% NaCl, pH 7.0. The retentate was collected and 0.2μm filtered and analyzed for protein content (Lowry or Micro-BCAcolorimetric assay), by SDS-PAGE, by amino acid analysis, and forimmunogenicity in mice.

EXAMPLE 3 Conversion by Capping of the Unreacted Bromoacetyl Groups toAminoacetyl Groups

Bromoacetylated CRM₁₉₇ (50 mg), prepared as described above in Example2, was transferred to a reaction vessel. To the stirred solution,maintained at 2-8° C., was added 1M sodium carbonate/bicarbonate.Titration was performed to achieve a target pH of 9.0, under argonatmosphere. Separately, 50 mg of Aβ peptide was weighed out anddissolved in WFI to 20 mg/mL. To this solution was added 1 M sodiumcarbonate/bicarbonate until pH 9.0 was attained. The peptide solutionwas added to the bromoacetylated CRM₁₉₇ solution, and the mixture wasstirred at 2-8° C. for 14-18 hours. The remaining bromoacetyl groupswere capped using 8% ammonium bicarbonate solution for 4 hours at 2-8°C.

The reaction mixture was 1.2 μm filtered into the retentate reservoir ofa UF/DF system (Millipore XL), and the conjugate was purified at roomtemperature by 30-fold diafiltration on a 10K or 30K MWCO membrane bydiafiltering vs 0.01 M sodium phosphate buffer/0.9% NaCl, pH 7.0. Theretentate was collected and 0.2 μm filtered and analyzed for proteincontent (Lowry or Micro-BCA calorimetric assay), by SDS-PAGE, by aminoacid analysis, and for immunogenicity in mice.

EXAMPLE 4 Quantitative Determination of S-Carboxymethylcysteine andS-Carboxymethylcysteamine as Evaluation of Degree of Conjugation andCapping of Peptide Immunogen-Protein/Polypeptide Conjugates

Acid hydrolysis of protein-peptide conjugates generated usingbromoacetyl activation chemistry resulted in the formation of acidstable S-carboxymethylcysteine (CMC) from the cysteines at theconjugated sites and the formation of acid stableS-carboxymethylcysteamine (CMCA) from the cysteamine at the capped sites(FIG. 2). All of the conjugated and capped lysines were converted backto lysine and detected as such. All other amino acids were hydrolyzedback to free amino acids except for tryptophan and cysteine, which weredestroyed by the hydrolysis conditions. Asparagine and glutamine wereconverted to aspartic acid and glutamic acid respectively.

Conjugate samples were diluted with deionized water to a total proteinconcentration of less then 1 mg/mL. Two 10 microgram aliquots of eachconjugate were dried and resuspended in 100 μL of 6N HCl [Pierce], 5 μLof melted phenol [Sigma-Aldrich], and 1 μL of 2-mercaptoethanol[Sigma-Aldrich]. The samples were then incubated under vacuum (100 mT)at 110° C. for 22 hours. The resulting hydrolysates were dried,resuspended in 250 μL of Beckman Na—S sodium citrate sample dilutionbuffer (pH 2.2) [Beckman Instruments, Inc., Fullerton, Calif.], andfiltered using Whatman 0.2 μM nylon syringe tip filters and 1 mLsyringes.

Each sample was then loaded into a Beckman 6300 amino acid analyzersample loop and placed in the analyzer. The amino acids of eachhydrolyzed sample and control were separated using ion exchangechromatography followed by reaction with Beckman Ninhydrin NinRXsolution at 135° C. The derivatized amino acids were then detected inthe visible range at 570 nm and 440 nm (see Table 1). A standard set ofamino acids [Pierce Amino Acid Standard H] containing 500 picomoles ofeach amino acid was run along with the samples and controls for each setof analysis. S-carboxymethylcysteine [Sigma-Aldrich] was added to thestandard.

TABLE 1 Retention Times for Amino Acids Using Gradient Program 1 on theBeckman 6300 Amino Acid Analyzer Wavelength Retention Time used for(min.) Amino Acid Detection 8.3 Carboxymethylcysteine CMC 570 9.6Aspartic Acid & Asparagine Asx 570 11.3 Threonine Thr 570 12.2 SerineSer 570 15.8 Glutamic Acid & Glutamine Glx 570 & 440 18.5 Proline Pro440 21.8 Glycine Gly 570 23.3 Alanine Ala 570 29.0 Valine Val 570 32.8Methionine Met 570 35.5 Isoleucine Ile 570 36.8 Leucine Leu 570 40.5Tyrosine Tyr 570 42.3 Phenylalanine Phe 570 45.4 CarboxymethylcysteamineCMCA 570 48.8 Histidine His 570 53.6 Lysine Lys 570 70.8 Arginine Arg570

The areas of each standard peak were used as a quantitative equivalencefor proportional evaluation of each sample. Proline was determined from440 nm and was converted to an equivalence in 570 nm using Glutamicacid, the closest amino acid.

Each of these picomole values was converted to a molar ratio of aminoacid residues using a comparison of picomoles of lysine to thetheoretical lysine value present in the protein. Lysine was chosen forthis evaluation based on its covalent attachment to Cysteine andCysteamine and the expected similar hydrolysis. The resulting numbers ofmoles of amino acids were then compared to the amino acid composition ofthe protein and reported along with the values for CMC and CMCA. The CMCvalue was used directly for evaluation of the degree of conjugation andthe CMCA value was used directly for evaluation of the degree ofcapping.

EXAMPLE 5 Characterization and Optimization of Aβ-CRM₁₉₇ PeptideConjugates

To verify conjugation, all peptide-CRM₁₉₇ conjugates were analyzed byamino acid analysis and matrix-assisted laser desorption ionization-timeof flight (MALDI-TOF) mass spectrometry. For each conjugate, the molesof peptide conjugated to each mole CRM₁₉₇ was determined by amino acidanalysis (number of S-carboxymethylcysteine residues) and MALDI-TOF massspectrometry. The values determined by each method were generally inagreement.

I. Size Exclusion Chromatography:

Batch concentrate samples were removed from storage and allowed to warmto room temperature. The Aβ peptide conjugate sample was gently mixed toinsure a homogeneous preparation. The Aβ peptide conjugate sample wasspun in an Eppendorf micro-centrifuge to remove any particulates. Thesupernatant was withdrawn for TosoHaas TSK-Gel G3000SW chromatography(TosoHaas, Stuttgart, Germany). A TosoHaas TSK-Gel G3000SW column wasconnected to a HPLC system and the pressure limit was set to 1.4 MPa.The column was equilibrated with at least 30 mL of PBS (10 mM sodiumphosphate, 150 mM NaCl, pH 7.2±0.1) at a flow rate of 0.75 mL/min. TheAβ peptide conjugate sample was loaded onto the TosoHaas TSK-Gel G3000SWcolumn using the following parameters:

-   -   Concentration of Aβ peptide conjugate sample: 1.5±1.0 mg/mL    -   Flow rate: 0.75 mL/min    -   Sample Volume: 0.1 mL    -   Run Time: 30 minutes

The absorbance was monitored at both 280 nm and 210 nm. For long termstorage, the TosoHaas TSK-Gel G3000SW column was equilibrated with atleast 50 mL of 20% ethanol at a flow rate of 0.5-1.0 mL/min.

II. PAGE (Polyacrylamide Gel Electrophoresis):

The activated (bromoacetylated) CRM₁₉₇ and the Aβ peptide-CRM₁₉₇conjugates were examined by SDS-Gels using a NuPAGE Bis-TrisElectrophoresis (Novex, Frankfurt, Germany) with a neutral pH, pre-castpolyacrylamide mini-gel system and NuPAGE MES SDS Running Buffer. An 8ug aliquot of each activated CRM or conjugate was mixed with reducingsample buffer and heated at 100° C. for 5 minutes. The conjugates andmolecular weight (MW) standards (Invitrogen, Carlsbad, Calif.) wereloaded on a 10% (w/v, acrylamide) NuPage gel (Novex) based upon aBis-Tris-HCl buffered system and run on MES SDS Running Buffer-PAGE(Laemmli). Following SDS-PAGE, the gel was stained with Pierce Gel CodeBlue (Pierce, Rockford, Ill.). Aβ peptide-CRM₁₉₇ conjugate wasrepresented by a major band around 66 kDa, above the band of native CRMand a dimer band around 120 kDa, along with minor multimer bands (datanot shown).

III. MALDI-TOF Mass Spectrometry Analysis of Peptide-CRM₁₉₇ Conjugates:

Mass spectrometry was used for immediate approximation of the degree ofconjugation. Suitable aliquots of activated CRM₁₉₇ and conjugate sampleswere analyzed by MALDI-TOF mass spectrometry using3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid) as the matrix.The molecular weight of activated CRM₁₉₇ determined by MALDI-TOF massspectrometry (Finnigan MAT Lasermat 2000 Mass Spectrometer, Ringoes,N.Y.) was found to be centered around 60.5 kDa and for conjugates variedfrom 65 kDa to 74 kDa depending on the degree of conjugation (data notshown). Up to 22 of the lysines (−50%) in CRM₁₉₇ were found to bemodified at 1:1 ratio.

IV. Optimization Experiments:

The degree of activation and conjugation are a function ofreagent:protein ratio, temperature of the reaction and pH of thereaction buffer. Some examples are given below to illustrate the optimalconjugation conditions carried out to identify the optimal pH conditionsin order to have reproducible process control parameters for conjugationreactions. Results (FIG. 3) showed that the conjugation reaction to Aβ5mer (DAEFRC) (SEQ ID NO:1) as well as Aβ 7mer (DAEFRHDC) (SEQ ID NO:2)is pH dependent and yields a higher degree of modification/conjugationwhen the pH of the reaction condition is increased. Using the TFA saltof 5mer and 7mer peptides, the degree of conjugation was evaluated at pH9.0 with varying amounts of peptide load (FIG. 4). It is evident fromthese results that peptide conjugates with a defined number of peptidecopies per CRM molecule can be generated by varying thepeptide/activated CRM ratio during the conjugation process. Similarexperiments were done using acetate salt of Aβ 7mer peptide.

For the Aβ1-7/CRM conjugation, the capping process was evaluated bycomparing the moles of CMCA per CRM to the moles of CMC per CRM. Sincethe total of the CMC and CMCA was constant for each peptide:CRM ratiotested, the capping process was presumed to be complete (FIG. 5). Thetotal modification in the conjugate stayed between 19 and 21, comparableto the number of lysines bromoacetylated (FIG. 5). These experimentswere done with TFA as the counterion for the peptide. The Aβ1-7/CRMconjugation was repeated using the acetate salt of the peptide ratherthan the TFA salt, and these data are shown in FIGS. 5 and 6. Thecapping process appeared to go to completion, with the total of the CMCand CMCA for each point staying between 20 and 22. The conditions forthe Aβ-CRM conjugation reaction have been optimized at pH 9.0, with thedegree of conjugation controlled by the peptide to CRM ratio in thereaction. By varying the ratio from 0.1 to 1.5, the degree ofconjugation can be varied (FIG. 6).

The degree of activation and conjugation are a function ofreagent:protein ratio, temperature of the reaction and pH of thereaction buffer. The degree of modification (conjugation) for eachconjugate was calculated by subtracting the mass value of activatedCRM₁₉₇ from the mass value of each conjugate and dividing by the mass ofthe peptide used to prepare the conjugate. The degree of modification(conjugation) for all of the conjugates is described in the Table 2.

The degree of conjugation was also compared to the values determined bythe estimated amount of S-carboxymethylcysteine residues formed per moleof CRM₁₉₇ (also shown in Table 2).

TABLE 2 Degree of Modification: Comparison of MALDI-TOF and AAA DataDegree of Degree of conjugation Da conjugation (From CMC-Amino (FromMass (From Mass Acid Sample Spectrometry) Spectrometry) Analysis) CRM₁₉₇58,408 — — BrAc-CRM 60,752 19 — Aβ1-7/CRM 74,463 14 15 Aβ1-7/CRM 72,37512 14 Aβ1-5/CRM 75,425 20 21 Aβ1-5/CRM 71,690 15 18

EXAMPLE 6 Immunogenicity Studies of Aβ Peptide Conjugates

Peptides spanning N-terminal residues 1-5, 1-7, 1-9, and 1-12 of Aβ(with and without the linker sequence GAGAC) and a peptide correspondingto the N-terminus of Aβ in reverse sequence from amino acid twelve toamino acid one (1-12mer in reverse sequence), each conjugated to CRM₁₉₇,were used to immunize mice along with an unconjugated Aβ 1-12 merpeptide in a formulation with STIMULON™ QS-21. Each group of mice wasimmunized subcutaneously with a dose of either 30 μg or 5 μg of one ofthe samples formulated with 20 μg of the adjuvant STIMULON™ QS-21, atthe beginning of the study (week 0) and subsequently at weeks 3 and 6.The study protocol is illustrated in Table 3.

As shown in Table 3, peptides spanning N-terminal residues 1-5, 1-7,1-9, and 1-12 of Aβ (with and without the linker sequence GAGAC) and apeptide corresponding to the N-terminus of Aβ in reverse sequence fromamino acid twelve to amino acid one (1-12mer in reverse) conjugated toCRM₁₉₇ were used to immunize mice along with unconjugated Aβ1-12 merpeptide in a formulation with QS-21. Each group of mice was vaccinatedsubcutaneously with a dose of either 30 μg or 5 μg of one of the samplesformulated with 20 μg of the adjuvant QS-21, at the beginning of thestudy (week 0) and subsequently at weeks 3 and 6. Swiss Webster micewere used for the entire study with 5 mice in each group. Injectionvolume=100 μl; B=Bleed; V=vaccinate; E=exsanguinate.

Anti-Aβ titers were measured by ELISA against Aβ and CRM₁₉₇ as describedbelow. Briefly, Costar 96 well plates (#3591) were coated overnight atroom temperature with 2 μg/mL Aβ1-42 in sterile carbonate/bicarbonatebuffer, pH 9.6. Plates were emptied and blocked for two hours at roomtemperature with 200 μl/well of 0.05% BSA in 1×PBS/0.05% Tween 20.Blocked plates were emptied and washed with a plate washer containingTBS, 0.1% Brij-35 (without azide) wash buffer. All primary antisera wereserially diluted with 0.05% BSA in 1×PBS containing 0.05% Tween 20/0.02%Azide and 100 μL of each dilution was then transferred to theappropriate plate wells and incubated at room temperature for 2 hours.Plates were then emptied/washed as described above. Alkaline phosphataseconjugated goat anti-mouse IgG secondary antibody from Southern Biotech(city, state) was diluted 1:1000 with 0.05% BSA in PBS containing 0.05%Tween 20/0.02% Azide and 100 μL was added to each well and incubated atroom temperature for 1 hour. Plates were then emptied/washed asdescribed above and finally incubated at room temperature for 1 hourwith 100 μL/well of a 1 mg/mL solution of p-nitrophenyl phosphatesubstrate prepared in diethanolamine/MgCl₂, pH 9.8. The colordevelopment was stopped with the addition of 50 μL/well of 3 N NaOH.Plates were read at 405 nM with a 690 nM reference. Endpoint titers werecalculated at an O.D. of 0.1 AU.

TABLE 3 Mouse Immunization Study Protocol Group Dose Code Description(μg) Wk 0 Wk 3 Wk 6 Wk 8 Wk 13 Wk 16 AE488 CRM/1-7 w/o linker 30 B, V B,V B, V B B E AE489 CRM/1-12 with 30 B, V B, V B, V B B E linker AE490CRM/1-9 with 30 B, V B, V B, V B B E linker AE491 CRM/1-7 with 30 B, VB, V B, V B B E linker AE492 CRM/1-5 w/o 30 B, V B, V B, V B B E linkerAE493 CRM/1-9 w/o 30 B, V B, V B, V B B E linker AE494 CRM/1-12 w/o 30B, V B, V B, V B B E linker AE495 CRM/1-5 with 30 B, V B, V B, V B B Elinker AE496 CRM/1-7 w/o 5 B, V B, V B, V B B E linker AE497 CRM/1-12with 5 B, V B, V B, V B B E linker AE498 CRM/1-9 with 5 B, V B, V B, V BB E linker AE499 CRM/1-7 with 5 B, V B, V B, V B B E linker AE500CRM/1-5 w/o 5 B, V B, V B, V B B E linker AE501 CRM/1-9 w/o 5 B, V B, VB, V B B E linker AE502 CRM/1-12 w/o 5 B, V B, V B, V B B E linker AE503CRM/1-5 with 5 B, V B, V B, V B B E linker AE504 CRM₁₉₇ C1-6151 30 B, VB, V B, V B B E AE505 CRM₁₉₇ C1-6151 5 B, V B, V B, V B B E AE506CRM/12-1mer 30 B, V B, V B, V B B E AE507 CRM/12-1mer 5 B, V B, V B, V BB E AE508 1-12mer peptide 30 B, V B, V B, V B B E AE509 1-12mer peptide5 B, V B, V B, V B B E AE510 Ab 30 B, V B, V B, V B B E AE511 Ab 5 B, VB, V B, V B B E

CRM₁₉₇ ELISA

Greiner 96 well plates (#650011) were coated at 37° C. for 90 minuteswith 5.0 μg/mL (100 μl/well) of CRM₁₉₇ in sterile carbonate/bicarbonatebuffer, pH 9.6. Plates were emptied and washed with a plate washercontaining 1×TBS, 0.1% Brij-35 wash buffer. All primary antisera wereserially diluted with 1×PBS containing 0.3% Tween 20/EDTA and 100 μL ofeach dilution was then transferred to the appropriate plate wells andincubated at 37° C. for 1 hour. The plates were then emptied/washed asdescribed above. Alkaline phosphatase conjugated goat anti-mouse IgGsecondary antibody from Southern Biotech was diluted 1:1000 with 1×PBScontaining 0.05% Tween 20/0.02% Azide and 100 μL was added to each welland incubated at 37° C. for 1 hour. Plates were then emptied/washed asdescribed above and finally incubated at room temperature for 1 hourwith 100 μL/well of a 1 mg/mL solution of p-nitrophenyl phosphatesubstrate prepared in diethanolamine/MgCl₂, pH 9.8. The development wasstopped with the addition of 50 μL/well of 3 N NaOH. Plates were read at405 nM with a 690 nM reference. Endpoint titers were calculated at anO.D. of 0.1 AU.

Tables 4-6 illustrate end point ELISA titers against Aβ. Followingprimary immunization, all eight conjugates (excluding the negativecontrol) induced measurable anti-Aβ IgG immune responses. However, the30 μg dose, but not the 5 μg dose, of Aβ gave a positive response atweek 3 following primary immunization. Among all the conjugates, itappears that Aβ 1-7 peptide conjugated without linker elicited as goodas or better response than other conjugates studied. At 5 μg dose,Aβ1-5C did better at weeks 8-16. Aβ1-7C was best at 30 μg dose. Analysisof antibody titers after second and third immunization with either 5 or30 μg dose indicate that the maximal immune response to Aβ for most ofthe conjugates was seen after the second immunization. At least in mice,the third immunization did not appear to enhance the immune response. Aβpeptide however, needed three immunizations with the 30 μg dose to reachmaximal immune response against the peptide (Table 5). In terms ofantibody decay over an extended period of time, the antibody level fromthe groups immunized with conjugates was reduced by 2 to 3 fold ascompared to the highest level within that group. Individual samples fromweeks 6 and 8 were analyzed to calculate GMTs against Aβ for each of thegroup (Table 6) to see if any conjugate group was substantially betterthan the others. Statistical analysis of week 6 titers from Aβ1-5C,Aβ1-7C and Aβ1-9C conjugates indicated that the Aβ1-7 conjugate induceda significantly higher titer. It is also evident from this experimentthat the linker sequence GAGAC did not contribute to enhancing theimmune response to the peptide.

TABLE 4 Table 4. Weeks 0, 3, 6, 8, 13, and 16 ELISA endpoint titersagainst Aβ using antiserum from 5 μg dose of peptide conjugates spanningvarying lengths of the N-terminus of Amyloid Aβ peptide. Ref: Elanhyperimmune polyclonal #592 = 3,073,307. Endpoint at O.D. 0.1 AU. SwissWebster mice were immunized SC-N with 5 μg of above antigens formulatedwith 20 μg STIMULON ™ QS-21 at weeks 0, 3, and 6. Group Week 3 Week 6Week 8 Week 13 Week 16 1-5C <100 14,960 687,691 882,012 625,208 771,8281-7C <100 51,253 1,280,181 860,463 520,060 571,043 1-9C <100 18,6151,008,872 622,325 348,967 380,755 1-12C <100 615 132,009 390,624 166,162184,170 1-5LC <100 4,999 458,075 454,631 237,573 220,091 1-7LC <10017,693 849,170 842,402 446,089 400,536 1-9LC <100 18,544 1,465,1151,180,347 571,127 579,477 1-12LC <100 12,664 908,360 598,867 368,101316,075 CRM₁₉₇ <100 <100 <100 <100 <100 <100 1-42 <100 <100 <100 <100<100 <100 1-12 <100 <100 <100 <100 <100 <100 12-1C <100 <100 <100 <100<100 <100

TABLE 5 Table 5. Weeks 0, 3, 6, 8, 13, and 16 ELISA endpoint titersagainst Aβ using antiserum from 30 μg dose of peptide conjugatesspanning varying lengths of the N-terminus of Amyloid Aβ peptide. Ref:Elan hyperimmune polyclonal #592 = 3,073,307. Endpoint at O.D. 0.1 AU.Swiss Webster mice were immunized SC-N with 30 μg of above antigensformulated with 20 μg STIMULON ™ QS-21 at weeks 0, 3, and 6. Group Week0 Week 3 Week 6 Week 8 Week 13 Week 16 1-5C <100 18,150 590,355 332,832204,645 176,159 1-7C <100 100,672 1,840,741 647,470 592,638 779,072 1-9C<100 18,520 1,184,696 713,494 363,459 327,065 1-12C <100 7,837 1,325,7251,126,389 681,268 577,604 1-5LC <100 16,347 469,191 184,077 177,358164,680 1-7LC <100 47,866 971,229 462,200 463,466 529,726 1-9LC <10059,002 921,544 787,273 405,023 500,468 1-12LC <100 27,348 697,150483,320 284,800 397,816 CRM₁₉₇ <100 <100 <100 <100 <100 <100 1-42 <100160 3,327 109,718 48,646 27,901 1-12 <100 <100 <100 <100 <100 <100 12-1C<100 <100 <100 <100 <100 <100

TABLE 6 Table 6. Weeks 6 and 8 ELISA endpoint GMTs against Aβ usingantisera from 30 μg dose of peptide conjugates spanning varying lengthsof the N-terminus of Amyloid-Aβ. Ref: Elan Hyperimmune Polyclonal #592 =3,073,307. Endpoint at O.D. 0.1 AU. Swiss Webster mice were immunizedSC-N with 30 μg of above antigens formulated with 20 μg STIMULON ™ QS-21at weeks 0, 3, and 6 Group Week 6 Week 8 1-5C   237,668 ^(a)   161,671^(b) 1-7C 1,866,702 ^(a )   881,146 ^(b) 1-9C   963,323 ^(a)   595,414^(b) 1-12C 940,260 955,470 1-5LC 395,553 141,084 1-7LC 516,921 394,5211-9LC 826,773 562,458 1-12LC 544,768 376,952 1-42    365  4,565 ^(a)Statistical analysis of week 6 titers from 1-5C, 1-7C, and 1-9C usingTukey-Kramer show a statistical difference between 1-5C vs 1-7C only,whereas, analysis using Student's T-test shows a statistical differencebetween 1-5C vs 1-7C and 1-5C vs 1-9C. ^(b) Statistical analysis of week8 titers from 1-5C, 1-7C, and 1-9C does not show a statisticaldifference among the three groups. However, there appears to be a trendthat may indicate a difference between 1-5C vs 1-7C.

PDAPP Mouse Brain Tissue Staining

The PDAPP brain tissue staining assay provides an indication of thefunctionality of the Aβ peptide conjugates and/or Aβ1-42 antiserum.Serum samples from individual mouse groups were separately analyzed fortheir ability to recognize PDAPP mouse brain tissue plaques containingamyloid peptide. The results are shown in Table 7A and 7B. With theexception of the Aβ 5mer conjugate antisera, there was a dose-relatedresponse in recognizing the plaques. Independent of the linker, 30 μgconjugate-induced antisera had better reactivity patterns as compared tothat of 5 μg conjugate antisera. However, with the Aβ 5mer conjugateantisera, there seems be similar or better reactivity for the 5 μggroup. Comparing all these results, it is concluded that conjugates madefrom Aβ1-5 mer through Aβ1-9 mer are sufficient in eliciting plaquesrecognizing immune response in mice and the presence of linker is notessential. The following conclusions can be drawn from this study: (a)All of the peptide conjugates induced high titered antiserum against thecarrier protein CRM₁₉₇ to equal or slightly higher levels as compared tothe unconjugated CRM₁₉₇ control (not shown). (b) The conjugates with theGAGAC linker did not enhance immunogenicity or functionality compared toconjugates without the linker. (c) The immunogenicity data and PDAPPbrain tissue staining (an initial indication of functional antibody)show that the Aβ1-5mer and Aβ1-7mer conjugates appeared to be thepreferred immunogens for further development.

TABLE 7A PDAPP mouse brain tissue staining. 5 μg Dose Without LinkerWith Linker PDAPP PDAPP Vaccine Animal # Staining Vaccine Animal #Staining CRM/Aβ 1-5 1 +(no diffuse) CRM/Aβ 1-5 1 − 2 ++/+++ 2 − 3 ++/+++3 ± 4 ++ 4 ± 5 ++ 5 ± CRM/Aβ 1-7 1 ++ CRM/Aβ 1-7 1 + 2 ++ 2 ++ 3 ++ 3 ++4 ++ 4 + 5 ++ 5 ++ CRM/Aβ 1-9 1 + CRM/Aβ 1-9 1 ++ 2 +/++ 2 ++ 3 ± 3 + 4± 4 + 5 ± 5 + CRM/Aβ 1-12 1 − CRM/Aβ 1-12 1 + 2 ? 2 + 3 ± 3 ++ 4 − 4 − 5± 5 ± CRM/Aβ 12-1mer 1 − Aβ42 1 − 2 − 2 − 3 ± 3 − 4 − 4 − 5 ± 5 −

All antiserum diluted 1:1000 for staining procedure.

TABLE 7B PDAPP mouse brain tissue staining. 30 μg Dose Without LinkerWith Linker PDAPP PDAPP Vaccine Animal # Staining Vaccine Animal #Staining CRM/Aβ 1-5 1 − CRM/Aβ 1-5 1 + 2 +/++ 2 − 3 − 3 − 4 ± 4 ± 5 ++ 5− CRM/Aβ 1-7 1 +/++ CRM/Aβ 1-7 1 + 2 ++ 2 ±/+ 3 ++ 3 +/++ 4 ++ 4 ±/+ 5++/+++ 5 +/++ CRM/Aβ 1-9 1 ++/+++ CRM/Aβ 1-9 1 +/++ 2 ++ 2 ++ 3 ++ 3 ++4 + 4 ± 5 + 5 +/++ CRM/Aβ 1-12 1 − CRM/Aβ 1-12 1 +/++ 2 +/++ 2 + 3 +/++3 − 4 ± 4 +/++ 5 ± 5 + CRM/Aβ 12-1mer 1 − Aβ 42 1 ± 2 − 2 − 3 − 3 − 4 −4 − 5 − 5 −

All antiserum diluted 1:1000 for staining procedure.

EXAMPLE 7 Immunogenicity Studies in Monkeys

Groups of 6 monkeys received 30 ug of 7mer conjugate (total conjugate)adjuvanted with either STIMULON™ QS-21, alum or RC529 SE formulation atdays 0, 29 and 58. Additional groups included were 30 ug 5mer conjugatewith either alum (Al(OH)₃) or RC529 SE, 75 and 300 μg of Aβ withSTIMULON™ QS-21 as positive controls. Positive controls were immunizedevery two weeks. At day 36 and 64 the anti-A

antibody titers were determined (FIGS. 7-9). On day 36, 7mer/CRMconjugates with STIMULON™ QS-21, Alum and RC529 SE elicited GMT titersof 10110, 13330 and 17090 respectively (FIG. 7). In contrast, Aβ1-42plus STIMULON™ QS-21 elicited GMTs of 223 and 1734 at 75 and 300 kg doselevels, respectively. The Aβ 5mer conjugate elicited a titer of 2134with alum and 15980 with RC529 SE. On day 64, i.e. after 3 doses ofconjugates with either STIMULON™ QS21 or RC-529 SE induced substantiallyhigher titers than post second dose (GMTs 69910 for 7 mer/RC-529 SE;21640 for Aβ 5mer/RC-529 SE and 30310 for Aβ 7mer/STIMULON™ QS-21) (FIG.8). Conjugates with alum elicited reduced titers at post thirdimmunization compared to post second immunization. It appears that theAβ 7mer conjugate elicited a better response as compared to the Aβ 5merconjugate. In monkeys, adjuvanting Aβ 7mer conjugate with RC-529 SE orSTIMULON™ QS-21 elicited the highest response (FIG. 9). The response tothe Aβ 7mer conjugate with alum was moderate and similar to that of 300ug Aβ1-42 with STIMULON™ QS-21.

Several conclusions can be drawn from the current example. First, bothconjugates are very immunogenic in primate species. Second, the presenceof adjuvants in the immunization formulation significantly influencesthe immune response. Third, except for the aluminum adjuvant, RC-529 SEand STIMULON™ QS-21 enhance the immune response after each dose ofimmunization at least up to three doses (FIG. 9). Overall, Aβ 7merconjugate induced higher antibody response in the presence of 529,followed by STIMULON™ QS-21 (see FIG. 9).

EXAMPLE 8 Preparation of Multiple Antigenic Peptide (Map) Conjugates andtheir Immunogenicity Study

Several methods are available for generating multiple antigenic sites onthe carriers. In the previous examples, each antigenic site isseparately conjugated to the carrier by defined conjugation and cappingchemistries. In this example, multiple antigenic sites are constructedby solid phase synthesis of tandem repeats of Aβ1-7 mer. Alternativelythese tandem repeats can be coupled with T-cell epitopes with or withoutlinking through a lysine core as described elsewhere. These multipleantigenic peptides were synthesized with an additional cysteinyl residuefor conjugation to the carrier protein. Peptides containing one repeatunit (1-7), three repeat units (1-7)₃ and five repeat units (1-7)₅ withan additional cysteinyl residue at the carboxyl end were synthesized.These peptides were covalently attached to bromoacetylated CRM overnightthrough their C-terminal cysteine residues. The reaction was carried outat pH 9.0-9.2 with peptide:CRM ratios added as outlined in Table 8.Bromoacetyl groups, which did not react with peptide, were capped withN-acetylcysteamine. These lots represent conjugates containing onesingle copy, three tandem copies, and five tandem copies of the Aβ1-7peptide conjugated to CRM, respectively. Table 8 briefly outlines theproperties of the samples.

TABLE 8 Multiple Antigenic Peptide (MAP) Conjugate Samples ConjugatePeptide: CRM (w/w) pH of reaction Ab(1-7)₁/CRM 0.37 8.99 Ab(1-7)₃/CRM1.02 8.95 Ab(1-7)₅/CRM 1.67 9.17

Peptide load (the average number of Aβ1-7 peptides per carrier) andcapping numbers (Table 9) are the numbers of unique amino acids (CMC orCMCA) per carrier as determined by amino acid analysis. The CMC and CMCAvalues were referenced to lysine.

TABLE 9 Degree of Conjugation and Capping of Each Conjugate CONJUGATEPeptide Load (CMC) Capping (CMCA) Aβ(1-7)₁/CRM 12.5 11.7 Aβ(1-7)₃/CRM10.4 15.2 Aβ(1-7)₅/CRM 9.8 15.9

Swiss-Webster mice (10 per group) were immunized subcutaneously with 1or 0.1 μg Aβ/CRM conjugated peptide. Half of the mice were immunizedwith the composition formulated with 100 μg of the adjuvant Al(OH)₃, andhalf were immunized without adjuvant. Immunizations were scheduled atweeks 0 and 3. Bleeds were scheduled for weeks 0, 3, and 6. Serumsamples were analyzed for antibody response against Aβ1-42 mer peptide.The results are shown in Table 10.

TABLE 10 Anti-Aβ Endpoint Titers for Multiple Antigenic Peptide (MAP)Conjugates Group Wk 0 Wk 3 Wk 6 Code Sample Description Adjuvant PoolGMT GMT AG332 1 μg Aβ (1-7)₁/CRM Al(OH)₃ <100 18,096 100,279 AG333 1 μgAβ (1-7)₃/CRM Al(OH)₃ <100 44,911 420,235 AG334 1 μg Aβ (1-7)₅/CRMAl(OH)₃ <100 27,032 394,488 AG335 0.1 μg Aβ (1-7)₁/CRM Al(OH)₃ <10019,350 66,834 AG336 0.1 μbg Aβ (1-7)₃/CRM Al(OH)₃ <100 13,307 208,272AG337 0.1 μg Aβ (1-7)₅/CRM Al(OH)₃ <100 1,196 22,665 AG338 1 μg Aβ(1-7)₁/CRM None <100 5,273 370,980 AG339 1 μg Aβ (1-7)₃/CRM None <1009,299 541,093 AG340 1 μg Aβ (1-7)₅/CRM None <100 3,100 185,272 AG341 0.1μg Aβ (1-7)₁/CRM None <100 340 25,839 AG342 0.1 μg Aβ (1-7)₃/CRM None<100 128 5,553 AG343 0.1 μg Aβ (1-7)₅/CRM None <100 668 2,098

All conjugates induced anti-Aβ1-42 antibody titer after primaryimmunization and the levels were substantially increased after thebooster dose. In the absence of aluminum adjuvant, the differences indose response were evident both at week 3 and week 6 bleeds. The higherdose elicited high-titered antibody response. Aluminum adjuvant elicitedsubstantially higher antibody response at week 3 at both dose levels(0.1 and 1 μg) as compared to the unadjuvanted groups. After secondaryimmunization, conjugates given at 1 μg dose elicited 5 to 10 foldincrease in antibody levels. At this dose level peptide conjugates with3 and 5 repeats induced higher antibody response than a single repeatcontaining conjugate. The titers against the CRM carrier were alsodetermined, and these are listed in Table 11.

TABLE 11 Anti-CRM Endpoint Titers for Multiple Antigenic Peptide (MAP)Conjugates Group Wk 0 Wk 3 Wk 6 Code Sample Description Adjuvant PoolGMT GMT AG332 1 μg Aβ(1-7)₁/CRM Al(OH)₃ <50 10,531 114,602 AG333 1 μgAβ(1-7)₃/CRM Al(OH)₃ <50 4,274 83,065 AG334 1 μg Aβ(1-7)₅/CRM Al(OH)₃<50 1,680 49,320 AG335 0.1 μg Aβ(1-7)₁/CRM Al(OH)₃ <50 1,114 13,231AG336 0.1 μg Aβ(1-7)₃/CRM Al(OH)₃ <50 197 1,484 AG337 0.1 μgAβ(1-7)₅/CRM Al(OH)₃ <50 65 222 AG338 1 μg Aβ(1-7)₁/CRM None <50 35 309AG339 1 μg Aβ(1-7)₃/CRM None <50 29 1,085 AG340 1 μg Aβ(1-7)₅/CRM None<50 29 542 AG341 0.1 μg Aβ(1-7)₁/CRM None <50 25 55 AG342 0.1 μgAβ(1-7)₃/CRM None <50 25 34 AG343 0.1 μg Aβ(1-7)₅/CRM None <50 29 NDAnimals were immunized at weeks 0 and 3 and bled at weeks 0, 3, and 6.Adjuvant: 100 μg Al(OH)₃ or none. ND = Not Determined.

Data in Table 11 indicates that the unadjuvanted groups induced very lowlevels of anti-CRM antibody response at both 1 μg as well as 0.1 μg doselevels even after two immunizations. However conjugates with aluminumhydroxide adjuvant induced substantial levels of anti-CRM antibodyresponse at 1 μg dose and much lower response at 0.1 μg dose. In thepresence of the adjuvant, CRM titers were highest for the single repeatconjugate, intermediate for the triple repeat conjugate, and lowest forthe quintuple repeat conjugate. This is as expected, since the CRM doseper peptide dose is lowest for Aβ(1-7)₅/CRM, and highest forAβ(1-7)₁/CRM. The differences were only statistically significant atweek 6 for the 0.1μμg dose.

The objective of the current invention is to elicit high titeredimmunogenic response against the antigenic hapten and not necessarilyagainst the carrier protein. Under certain circumstances it is desirableto elicit optimal immune response against the hapten antigenicdeterminant with least or no immune response against the carrierprotein. For such applications, conjugates with tandem repeats ofmultiple antigenic determinants with unadjuvanted formulation will servethe need.

EXAMPLE 9 Preparation of Aβ-Peptide Conjugates with Various CarrierProteins and their Immunogenicity

This example compares the immunogenicity of conjugates using sixdifferent carrier proteins. The acetate salt of Aβ1-7 was added tobromoacetylated carriers in a 1:1 ratio by weight at pH 9. Allconjugates except Aβ1-7/rC5ap were capped with N-acetylcysteamine. Allof the alternative carriers are recombinant bacterial proteins,including CRM (diphtheria toxoid), recombinant C5a peptidase (rC5ap;cloned from Streptococcus agalactiae, includes D130A and S512Amutations), ORFs 1224, 1664, 2452 (all cloned from Streptococcuspyogenes), and T367, T858 (each cloned from Chlamydia pneumoniae). Asummary of the carriers used is found in Table 12. The degree ofconjugation and capping of each Aβ1-7 conjugate to these carriers arepresented in Table 13.

This study showed that the recombinant C5a peptidase conjugate inducedhigher titers against Aβ than most of the other carriers tested,including CRM. This difference was statistically significant for week 6titers of groups that received aluminum hydroxide. In addition, theAβ1-7/T858 conjugate was significantly more immunogenic than most otherconjugates in the absence of adjuvant. The only conjugate that performedpoorly relative to the CRM control conjugate was Aβ1-7/T367, a conjugatethat also did not react with an Aβ specific monoclonal antibody byWestern blot. This study confirms that numerous other carriers can besuccessfully used to immunize against the Aβ peptide.

TABLE 12 List of Carriers and Conjugate Properties CARRIER PROTEIN MW ofcarrier (Da) # of lysines CRM 58,408 39 rC5ap 108,560 85 ORF1224 30,95018 ORF1664 31,270 38 ORF2452 31,790 29 T367 49,700 29 T858 37,190 23

TABLE 13 Degree Of Conjugation and Capping of Each Conjugate CappingCONJUGATE Peptide load (CMC) (CMCA) Aβ1-7/rC5ap 25.9 — Aβ1-7/ORF122412.8 5.7 Aβ1-7/ORF1664 13.4 10.8 Aβ1-7/ORF2452 12.03 10.5 Aβ1-7/T36713.2 8.2 Aβ1-7/T858 5.2 1.7Conjugation results: Peptide load (the average number of Aβ1-7 peptidesper carrier) and capping number are the numbers of unique amino acids(CMC or CMCA) per carrier as determined by amino acid analysis. The CMCand CMCA values were referenced to lysine.

Immunization Results

The geometric mean titer for each group in this study is listed in Table14. At week 3, regardless of the presence of adjuvant, Aβ1-7/rC5apinduced significantly higher anti-Aβ titers than the correspondingconjugates prepared with Streptococcus pyogenes ORFs 1224, 1664, 2452,or Chlamydia pneumoniae ORFs T367 and T858. At week 3 in the absence ofadjuvant, Aβ1-7/rC5ap was also more immunogenic than all otherconjugates except Aβ1-7/T858. The T858 conjugate without Al(OH)₃ inducedhigher titers than the ORF1224, ORF1664, ORF2452, and CRM conjugateswithout adjuvant. The only conjugate that was significantly lessimmunogenic than Aβ1-7/CRM was Aβ1-7/T367 (p<0.00002). The T367 carrierperformed poorly with or without adjuvant at both weeks 3 and 6. At week6, the rC5ap conjugate with aluminum hydroxide was more immunogenicp<0.04) than all the other conjugates except Aβ1-7/ORF2452. In theabsence of adjuvant, both Aβ1-7/rC5ap and Aβ1-7/T858 inducedsignificantly higher titers than the ORF1224, ORF1664, or T367conjugates. Aβ1-7/CRM without aluminum hydroxide induced higher titersthan either Aβ1-7/ORF1664 or Aβ1-7/T367.

TABLE 14 Anti-Aβ1-42 Endpoint Titers. GROUP SAMPLE WK 0 WK 3 WK 6 CODEDESCRIPTION ADJUVANT POOL GMT GMT AG344 5 μg Aβ1-7/CRM Al(OH)₃ <10021,404 54,157 AG345 5 μg Aβ1-7/rC5ap Al(OH)₃ <100 61,967 402,972 AG346 5μg Aβ1-7/ORF1224 Al(OH)₃ <100 10,711 30,084 AG347 5 μg Aβ1-7/ORF1664Al(OH)₃ <100 7,188 43,226 AG348 5 μg Aβ1-7/ORF2452 Al(OH)₃ <100 11,437109,091 AG349 5 μg Aβ1-7/T367 Al(OH)₃ <100 321 5,139 AG350 5 μgAβ1-7/T858 Al(OH)₃ <100 16,656 33,328 AG351 5 μg Aβ1-7/CRM None <1002,615 119,488 AG352 5 μg Aβ1-7/rC5ap None <100 11,858 279,113 AG353 5 μgAβ1-7/ORF1224 None <100 1,674 18,719 AG354 5 μg Aβ1-7/ORF1664 None <100119 9,832 AG355 5 μg Aβ1-7/ORF2452 None <100 2,493 76,038 AG356 5 μgAβ1-7/T367 None <100 50 620 AG357 5 μg Aβ1-7/T858 None <100 28,820275,202Animals were immunized at weeks 0 and 3 and bled at weeks 0, 3, and 6.Dose is based on the total amount of conjugate. Adjuvant: 100 μg Al(OH)₃or none.

EXAMPLE 10 Preparation of Additional Aβ Peptide-Protein Conjugates I.Activation

Thawed CRM₁₉₇ (8 mL, 59.84 mg, at 7.48 mg/mL) was dissolved in 0.1 Mborate buffer (pH 9, 3.968 mL) to bring the concentration to 5 mg/mL.The solution was cooled in an ice bath to 0-5° C. Bromoacetic acidN-hydroxysuccinimide (59.9 mg) (Aldrich-Sigma) was dissolved in DMF (100μL) (Aldrich-Sigma) and added dropwise, to the solution of CRM₁₉₇. Uponaddition of the bromoacetic acid N-hydroxysuccinimide, a precipitate wasobserved. When the pH was checked, it decreased to pH 6. The pH of thereaction mixture was brought back to pH 9 by adding more 0.1 M boratebuffer. Reaction mixture was then stirred at 4° C. for 1 hr, with gentleswirling. The mixture was purified and concentrated using YM-10centriprep centrifugal concentration and repurified on Sephadex G-25using 10 mM borate as the eluent. Fractions positive to Bradford Reagentwere pooled and concentrated using centriprep YM-10. The degree ofbromoacetylation was determined by Bradford assay (linear). Theconcentration was found to be 5.36 mg/mL (Yielded 30 mg). The finalconcentration was then adjusted to be 5 mg/mL and was stored in thefreezer in 5% sucrose until further use.

II. Conjugation

For each conjugation, thawed bromoacetylated CRM₁₉₇ was used. Peptideswere dissolved in borate buffer (2.5 mg in 125 ml of 0.1 M boratebuffer). Slight insolubility was observed with Aβ peptides KLVFFAEDC(SEQ ID NO:45), CLVFFAEDV (SEQ ID NO:47), CKLVFFAED (SEQ ID NO:48), andLVFFAEDC (SEQ ID NO:50). Bromoacetylated CRM₁₉₇ (5 mg/mL) was treatedwith the peptide solutions/suspensions. The ratio of the peptide andprotein in the mixture was 1:2. Turbidity was observed in the conjugatemixtures with peptides KLVFFAEDC (SEQ ID NO:45), CLVFFAEDV (SEQ IDNO:47), CKLVFFAED (SEQ ID NO:48), and KLVFFAEDC (SEQ ID NO:45). Themixtures were then checked for pH (pH 9) and incubated at 4° C.overnight with slow swirling. Final concentrations of the mixtures weremade to 3 mg/mL before incubation. The turbidity of the conjugatemixtures with peptides CLVFFAEDV (SEQ ID NO:47) and LVFFAEDC (SEQ IDNO:50) disappeared after incubation. However, KLVFFAEDC (SEQ ID NO:45)and CKLVFFAED (SEQ ID NO:48) were still slightly turbid. Soluble mockprotein conjugate was also prepared with cysteamine at a ratio of 1:1(w/w). Synthesized peptides were obtained from BIOSOURCE with about 95%purity.

Octamers: LVFFAEDVC (SEQ ID NO:44) KLVFFAEDC (SEQ ID NO:45) VFFAEDVGC(SEQ ID NO:43) CLVFFAEDV (SEQ ID NO:47) CKLVFFAED (SEQ ID NO:48)CVFFAEDVG (SEQ ID NO:46) Heptamers: VFFAEDVC (SEQ ID NO:49) LVFFAEDC(SEQ ID NO:50)

III. Capping Unreacted Lysine Groups on Protein:

The unreacted lysines were capped with N-acetylcysteamine (CMCA;Aldrich-Sigma) at a ratio of 1/1 (w/w) for 4 hr at 4° C. while swirlingin the dark. The unreacted peptides and capping reagents were removedfrom the conjugates by dialysis using Slide-A-Lyzer cassette (M, cut off10,000) (Pierce) against PBS buffer (2 L) overnight (13 hr). Bufferexchange and dialysis was done twice (2×14 hr). Slight insolubility wasobserved in the conjugates with peptides KLVFFAEDC (SEQ ID NO:45) andCKLVFFAED (SEQ ID NO:48). All conjugates were then stored in therefrigerator at 4° C. in a perservative.

IV. Characterization of the Protein Carrier:

MALDI-TOF MS was used to determine the mass of bromoacetylated CRM₁₉₇and the mass of the mock conjugate N-acetylcysteamine-CRM₁₉₇.

Based on the masses of the CRM₁₉₇ and bromoacetylated CRM₁₉₇, 11 lysineresidues were modified.

(59941.46−58590.29)/122=11

Where;

-   -   Mw of CRM₁₉₇ is 58624.29    -   Mw of bromoacetylated CRM₁₉₇ is 59941.46    -   Mw of bromoacetate is 122

The degree of bromoacetylation was more than 28%. (The total number oflysines in CRM₁₉₇ was 39). From these 11 modified lysine residues, 10were coupled with cysteamine. The coupling efficiency was 90%.

(61143−59941)/119=10

Where;

-   -   Mw of bromoacetylated CRM₁₉₇ is 59941.46    -   Mw of mock conjugate is 61143    -   Mw of the N-acetylcysteamine is 119

(10/11)×100=90

V. Characterization of the Peptide-Protein Conjugates by SDS-PAGEWestern Blot Analysis with Tris-Tricine Precast Gel:

The protein-peptide conjugates were analyzed by Western blot. The lanesare: marker (lane 1); L-28375 24/01 (lane 2); L-28375 24/02 (lane 3);L-28375 24/03 (lane 4); L-28375 24/04 (lane 5); L-28375 24/05 (lane 6);L-28375 24/06 (lane 7) L-28375 24/07 (lane 8); L-28375 24/08 (lane 9);L-28375 24/09 (Mock) (lane 10); and, BrAcCRM₁₉₇ (lane 11). A peptidespecific monoclonal antibody from mice (248-6H9-806 Aβ17-28) was used asthe primary antibody (antisera) (1:3000 dilution was found to be thebest). Goat-Anti mouse IgG (H+ L)-HPR was the secondary antibody (1:1000dilution). It was observed that all the conjugates were recognized bythe primary antibody, except for the mock conjugate and the activatedCRM₁₉₇. (See FIG. 10.)

Protein Concentration

Protein concentrations of the conjugate samples were determined by thePierce BCA assay. (See Table 15.)

Amino Acid Analysis

Amino acid analysis was carried out to determine the degree ofconjugation. T degree of conjugation was calculated based on the CMCA(carboxymethylcycteamine) residues found in the conjugates. CMCA wasused to cap the unreacted activated sites after conjugation with thepeptides. (See Table 15.)

TABLE 15 Degree of Conjugation of Peptides with BrAcCRM₁₉₇ Final Degreeof Peptide Sequence Concentration Conjugation Conjugate Code (SEQ IDNO:) (mg/mL) (Based on CMCA) L-28375 24/01 LVFFAEDV-C 1.67 8/10 (SEQ IDNO: 44) L-28375 24/02 KLVFFAED-C 0.82 5/10 (SEQ ID NO: 45) L-28375 24/03VFFAEDVG-C 1.43 8/10 (SEQ ID NO: 43) L-28375 24/04 C-LVFFAEDV 1.04 9/10(SEQ ID NO: 47) L-28375 24/05 C-KLVFFAED 0.78 1/10 (SEQ ID NO: 48)L-28375 24/06 C-VFFAEDVG 0.97 9/10 (SEQ ID NO: 46) L-28375 24/07VFFAEDV-C 1.00 7/10 (SEQ ID NO: 49) L-28375 24/08 LVFFAED-C 0.99 8/10(SEQ ID NO: 50) L-28375 24/09 1.89 10/11  (Mock)

All colorimetric assays were performed using microplatespectrophotometer and SOFTmax Pro.

EXAMPLE 11 Immunogenic Studies of Aβ Peptide Conjugates in Swiss WebsterMice

Outbred Swiss Webster mice were immunized with VFFAEDVG-C (SEQ IDNO:43), LVFFAEDV-C (SEQ ID NO:44), KLVFFAED-C (SEQ ID NO:45), C-VFFAEDVG(SEQ ID NO:46), C-LVFFAEDV (SEQ ID NO:47), C-KLVFFAED (SEQ ID NO:48),VFFAEDV-C (SEQ ID NO:49), LVFFAED-C (SEQ ID NO:50) each conjugated toCRM₁₉₇, or with Aβ1-7CRM₁₉₇, all formulated with the adjuvant RC 529 SE.Nine groups of 10 animals per group were immunized subcutaneously withone of the Aβ peptide conjugates at the beginning of the study (week 0)and subsequently at week 4. Serum was collected prior to, but on thesame days as immunization.

Immunogenic Studies of Aβ Peptide Conjugates in Inbred Balb/c Mice

Inbred Balb/c mice were immunized as in the preceding paragraph, butwere also boosted with conjugate and adjuvant at week 12.

Results

Sera from both studies are being collected for analysis of Aβ₁₃₋₂₈peptide-specific IgG antibody titer. Sera from Balb/c mice are alsocollected for analysis one day prior to the week 12 boost, and one weekthereafter. Spleen cells from animals used in Example 11 are evaluatedfor their potential to respond in-vitro to stimulation with anoverlapping pool of peptides spanning Aβ₁₋₄₂, fall length Aβ₁₋₄₂,CRM₁₉₇, or polyclonal activators. Analysis is comprised of Elispotreadout for interleukins 4 and 5, and interferon-gamma. Upon completion,the Aβ peptide conjugates are be evaluated as described above and asdescribed in Example 6.

EXAMPLE 12 Immunogenic Studies of Aβ Peptide Conjugates in PSAPP Mice

PSAPP mice are immunized with VFFAEDVG-C (SEQ ID NO:43), LVFFAEDV-C (SEQID NO:44), KLVFFAED-C (SEQ ID NO:45), C-VFFAEDVG (SEQ ID NO:46),C-LVFFAEDV (SEQ ID NO:47), C-KLVFFAED (SEQ ID NO:48), VFFAEDV-C (SEQ IDNO:49), LVFFAED-C (SEQ ID NO:50). The PSAPP mouse, a doubly transgenicmouse (PSAPP) overexpressing mutant APP and PS1 transgenes, is describedin Holcomb, et al. (1998) Nature Medicine 4:97-11.

Immunogenic Studies of Aβ Peptide Conjugates in PDAPP Mice

PDAPP mice are immunized with VFFAEDVG-C (SEQ ID NO:43), LVFFAEDV-C (SEQID NO:44), KLVFFAED-C (SEQ ID NO:45), C-VFFAEDVG (SEQ ID NO:46),C-LVFFAEDV (SEQ ID NO:47), C-KLVFFAED (SEQ ID NO:48), VFFAEDV-C (SEQ IDNO:49), LVFFAED-C (SEQ ID NO:50) The PDAPP mouse expresses a mutant formof human APP (APP^(V71F)) and develops Alzheimer's disease at a youngage (Bard, et al. (2000) Nature Medicine 6:916-919; Masliah E, et al.(1996) J Neurosci. 15; 16(18):5795-811).

Results

Sera from both studies are collected for analysis of Aβ₁₃₋₂₈peptide-specific IgG antibody titer. Upon completion, the Aβ peptideconjugates will be evaluated as described above and as described inExamples 6 and 11, as well as in the contextual fear conditioning (CFC)assay.

Contextual fear conditioning is a common form of learning that isexceptionally reliable and rapidly acquired in most animals, forexample, mammals. Test animals learn to fear a previously neutralstimulus and/or environment because of its association with an aversiveexperience. (see, e.g., Fanselow, Anim. Learn. Behav. 18:264-270 (1990);Wehner et al., Nature Genet. 17:331-334. (1997); Caldarone et al.,Nature Genet. 17:335-337 (1997)).

Contextual fear conditioning is especially useful for determiningcognitive function or dysfunction, e.g., as a result of disease or adisorder, such as a neurodegenerative disease or disorder, an Aβ-relateddisease or disorder, an amyloidogenic disease or disorder, the presenceof an unfavorable genetic alteration effecting cognitive function (e.g.,genetic mutation, gene disruption, or undesired genotype), and/or theefficacy of an agent, e.g., an Aβ conjugate agent, on cognitive ability.Accordingly, the CFC assay provides a method for independently testingand/or validating the therapeutic effect of agents for preventing ortreating a cognitive disease or disorder, and in particular, a diseaseor disorder affecting one or more regions of the brains, e.g., thehippocampus, subiculum, cingulated cortex, prefrontal cortex, perirhinalcortex, sensory cortex, and medial temporal lobe.

Typically, the CFC assay is performed using standard animal chambers andthe employment of conditioning training comprising a mild shock (e.g.,0.35 mA foot shock) paired with an auditory (e.g., a period of 85 dbwhite noise), olfactory (e.g., almond or lemon extract), touch (e.g.,floor cage texture), and/or visual cue (light flash). The response tothe aversive experience (shock) is typically one of freezing (absence ofmovement except for respiration) but may also include eye blink, orchange in the nictitating membrane reflex, depending on the test animalselected. The aversive response is usually characterized on the firstday of training to determine a baseline for unconditioned fear, withaversive response results on subsequent test days, e.g., freezing inpresence of the context and/or cue but in the absence of the aversiveexperience, being characterized as context and cue conditioned fear,respectively. For improved reliability, test animals are typicallytested separately by independent technicians and scored over time.Additional experimental design details can be found in the art, forexample, in Crawley, J N, What's Wrong with my Mouse; BehavioralPhenotyping of Transgenic and Knockout Mice, Wiley-Liss, NY (2000).

Exemplary test animals (e.g., model animals) include mammals (e.g.rodents or non-human primates) that exhibit prominent symptoms orpathology that is characteristic of an amyloidogenic disorder such asAlzheimer's. Model animals may be created by selective inbreeding for adesired or they may genetically engineered using transgenic techniquesthat are well-known in the art, such that a targeted genetic alteration(e.g. a genetic mutation, gene disruption) in a gene that is associatedwith the dementia disorder, leading to aberrant expression or functionof the targeted gene. For example, several transgenic mouse strains areavailable that overexpress APP and develop amyloid plaque pathologyand/or develop cognitive deficits that are characteristic of Alzheimer'sdisease (see for example, Games et al., supra, Johnson-Wood et al.,Proc. Natl. Acad. Sci. USA 94:1550 (1997); Masliah E and Rockenstein E.(2000) J Neural Transm Suppl.; 59: 175-83).

Alternatively, the model animal can be created using chemical compounds(e.g. neurotoxins, anesthetics) or surgical techniques (e.g.stereotactic ablation, axotomization, transection, aspiration) thatablate or otherwise interfere with the normal function of an anatomicalbrain region (e.g. hippocampus, amygdala, perirhinal cortex, medialseptal nucleus, locus coeruleus, mammalary bodies) or specific neurons(e.g. serotonergic, cholinergic, or dopaminergic neurons) that areassociated with characteristic symptoms or pathology of theamyloidogenic disorder. In certain preferred embodiments, the animalmodel exhibits a prominent cognitive deficit associated with learning ormemory in addition to the neurodegenerative pathology that associatedwith a amyloidogenic disorder. More preferably, the cognitive deficitprogressively worsens with increasing age, such that the diseaseprogression in the model animal parallels the disease progression in asubject suffering from the amyloidogenic disorder.

Contextual fear conditioning and other in vivo assays to test thefunctionality of the conjugates described herein may be performed usingwild-type mice or mice having a certain genetic alteration leading toimpaired memory or mouse models of neurodegenerative disease, e.g.,Alzheimer's disease, including mouse models which display elevatedlevels of soluble Aβ in the cerebrospinal fluid (CSF) or plasma. Forexample, animal models for Alzheimer's disease include transgenic micethat overexpress the “Swedish” mutation of human amyloid precursorprotein (hAPPswe; Tg2576) which show age-dependent memory deficits andplaques (Hsiao et al (1996) Science 274:99-102). The in vivofunctionality of the conjugates described herein can also be testedusing the PS-1 mutant mouse, described in Duff, et al. (1996) Nature383, 710-713. Other genetically altered transgenic models of Alzheimer'sdisease are described in Masliah E and Rockenstein E. (2000) J NeuralTransm Suppl. 59:175-83.

In various aspects, the methods of the invention comprise theadministration of an Aβ conjugate that is capable of improving cognitionin a subject wherein the Aβ conjugate has been identified in using anassay which is suitably predictive of immunotherapeutic efficacy in thesubject. In exemplary embodiments, the assay is a model animal assaythat is based, at least in part, on comparing cognition, as determinedfrom a contextual fear conditioning study, of an animal afteradministration of a test immunological reagent to the animal, ascompared to a suitable control. The CFC assay evaluates changes incognition of an animal (typically a mouse or rat) upon treatment with apotential therapeutic compound. In certain embodiments, the change incognition evaluated is an improvement in memory impairment status or areversal of memory deficit. Accordingly, the CFC assay provides a directmethod for determining the therapeutic effect of agents for preventingor treating cognitive disease, and in particular, a disease or disorderaffecting one or more regions of the brains, e.g., the hippocampus,subiculum, cingulated cortex, prefrontal cortex, perirhinal cortex,sensory cortex, and medial temporal lobe. Such CFC assays are discussedin copending U.S. Patent Application Ser. No. 60/______ entitled“Contextual Fear Conditioning for Predicting Immunotherapeutic Efficacy”(bearing Attorney Docket No. ELN-058-1), filed on Dec. 15, 2004, andU.S. Patent Application Ser. No. 60/______ entitled “Contextual FearConditioning for Predicting Immunotherapeutic Efficacy” (bearingAttorney Docket No. ELN-058-2) the entire contents of which are herebyincorporated by reference.

1-379. (canceled)
 380. An immunogenic conjugate generated according to amethod comprising the steps of: (a) introducing a reactive group into anamino acid residue of a peptide immunogen wherein the peptide immunogenis an Aβ peptide or a fragment or analog thereof; (b) derivatizing oneor more functional groups of a carrier protein to generate an activatedfunctional group on the carrier protein; (c) reacting the peptideimmunogen of step (a) with the carrier protein of step (b) underconditions to form a conjugate, wherein the reactive group of thepeptide immunogen is covalently attached to the activated functionalgroup on the carrier protein; and (d) further reacting the conjugate ofstep (c) with a capping reagent to inactivate any remaining activatedfunctional group on the carrier protein to generate the immunogenicconjugate; and wherein the immunogenic conjugate has the formula:

wherein, C is a carrier protein and X^(d) is a derivatized functionalgroup of an amino acid residue of the carrier protein, and wherein P isa peptide immunogen comprising Aβ peptide or fragments of Aβ or analogsthereof covalently attached via a reactive group of an amino acidresidue of the peptide immunogen to the derivatized functional group ofthe amino acid residue of the carrier protein, R is a capping moleculecovalently attached to the derivatized functional group of an amino acidresidue of the carrier protein, which preserves the functionality of thecarrier such that it retains its ability to elicit the desired immuneresponse against the peptide immunogen that would otherwise not occurwithout a carrier, n is an integer greater than 0, but less than orequal to 85, and p is an integer greater than 0, but less than
 85. 381.The immunogenic conjugate of claim 380 wherein the carrier protein isselected from the group consisting of human serum albumin, keyholelimpet hemocyanin (KLH), immunoglobulin molecules, thyroglobulin,ovalbumin, influenza hemagglutinin, PADRE polypeptide, malariacircumsporozite (CS) protein, hepatitis B surface antigen (HBSAg19-28),Heat Shock Protein (HSP) 65, Mycobacterium tuberculosis, cholera toxin,cholera toxin mutants with reduced toxicity, diphtheria toxin, CRM₁₉₇protein that is cross-reactive with diphtheria toxin, recombinantStreptococcal C5a peptidase, Streptococcus pyogenes ORF1224,Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF2452,Streptococcus pneumoniae pneumolysin, pneumolysin mutants with reducedtoxicity, Chlamydia pneumoniae ORF T367, Chlamydia pneumoniae ORF T858,Tetanus toxoid, HIV gp120 T1, components recognizing microbial surfaceadhesive matrix molecules (MSCRAMMS), growth factors, hormones,cytokines and chemokines.
 382. The immunogenic conjugate of claim 381,wherein the carrier protein is CRM₁₉₇.
 383. The immunogenic conjugate ofclaim 380, wherein the peptide immunogen is an Aβ fragment.
 384. Theimmunogenic conjugate of claim 383, wherein the Aβ fragment is selectedfrom the group consisting of Aβ1-3, 1-4, 1-5, 1-6, 1-7, 1-9, 1-10, 1-11,1-12, 1-16, 1-28 3-6, 3-7, 13-28, 15-24, 16-22, 16-23, 17-23, 17-24,18-24, 18-25, 17-28, 25-35, 33-42, 35-40, and 35-42.
 385. Theimmunogenic conjugate of claim 380, wherein the step of introducing thereactive group into the peptide immunogen comprises adding an amino acidresidue having the reactive group.
 386. The immunogenic conjugate ofclaim 385, wherein the amino acid residue is a cysteine residue in whichthe reactive group comprises —SH, or the amino acid residue is anarginine residue and the reactive group comprises a guanidyl group, orthe amino acid residue is a glutamate or aspartate residue and thereactive group comprises —COOH, or the amino acid residue is a lysineresidue and the reactive group comprises —NH₂.
 387. The immunogenicconjugate of claim 384, wherein the reactive group introduced into thepeptide immunogen comprises a —SH of a cysteine residue, and wherein thecysteine residue is introduced into the Aβ peptide immunogen by additionduring peptide synthesis.
 388. The immunogenic conjugate of claim 387,wherein the cysteine residue is localized at the carboxy-terminus of theAβ peptide immunogen.
 389. The immunogenic conjugate of claim 380,wherein the step of introducing the reactive group into the peptideimmunogen comprises generating a pendant thiol group on an amino acidresidue susceptible to such modification via a thiolating reagent. 390.The immunogenic conjugate of claim 389, wherein the thiolating reagentcomprises N-acetylhomocysteine thiolactone.
 391. The immunogenicconjugate of claim 385, wherein the amino acid residue is localized atthe amino-terminus of the Aβ peptide immunogen or at carboxy-terminus ofthe Aβ peptide immunogen.
 392. The immunogenic conjugate of claim 389,wherein the amino acid residue is localized at the amino-terminus of theAβ peptide immunogen or at carboxy-terminus of the Aβ peptide immunogen.393. The immunogenic conjugate of claim 380, wherein the carrier proteinfurther comprises one or more polypeptide linkers covalently attached tothe carrier protein, and wherein the one or more functional groupscomprise a substituent of the one or more polypeptide linkers.
 394. Theimmunogenic conjugate of claim 380, wherein the reactive group of theamino acid residue of the peptide immunogen comprises a free sulfhydrylgroup.
 395. The immunogenic conjugate of claim 393, wherein the freesulfhydryl group is a side chain of a cysteine residue or a thiolatedside chain of a lysine residue.
 396. The immunogenic conjugate of claim380, wherein the reactive group of the amino acid residue of the peptideimmunogen comprises a guanidyl group, a carboxyl group, or an ε-aminogroup.
 397. The immunogenic conjugate of claim 384, wherein the reactivegroup of the amino acid residue comprises a free sulfhydryl group of acysteine residue.
 398. The immunogenic conjugate of claim 396, whereinthe cysteine residue is localized at the carboxy-terminus of the peptideimmunogen.
 399. The immunogenic conjugate of claim 380, wherein thecarrier protein further comprises a peptide linker covalently attachedto the carrier protein, and wherein the functional group comprises asubstituent of the peptide linker.